dna replication
TRANSCRIPT
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DNA ReplicationJason Ryan, MD, MPH
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DNA
• Contains genetic code
• Nucleus of eukaryotic cells
• Cytoplasm of prokaryotic cells
• Replicated for cell division/growth
Wikipedia/Public Domain
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DNA Structure
1. Sugar (ribose) backbone
2. Phosphate
3. Nitrogenous base
Wikipedia/Public Domain
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Base Pairing
• DNA• Adenine-Thymine
• Guanine-Cytosine
• RNA• Adenine-Uracil
• Guanine-Cytosine
• Antiparallel
Wikipedia/Public Domain
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Nucleotides
• Synthesized as monophosphates
• Converted to triphosphate form
• Triphosphate form added to DNA
Deoxy-adenosine Triphosphate
5’
3’
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DNA Replication
Thymidine
Adenosine
Guanosine
5’
5’
3’
3’
Cytosine
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DNA Replication
Thymidine
Adenosine
Guanosine
5’ 3’
5’3’
Cytosine
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DNA Replication
Thymidine
Adenosine
Guanosine
5’
5’
3’
3’
5’
5’
3’
3’
Cytosine
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DNA Replication
5’
5’
3’
3’
Originof
Replication
DNA Helicase
ATP
ssBP
ssBP
ssBP
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DNA Replication
• Helicase• Unwinds/opens double helix
• Hydrolyzes ATP
• Single strand binding proteins• Assist helicase
• Stabilize and straighten single strands of DNA
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Origin of Replication
• Specific DNA sequences• Attract initiator proteins
• Easy to unwind/open
• Fewer bonds A-T• “AT rich” sequences
• Easy to open
Wikipedia/Public Domain
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DNA Polymerases
• Bacteria (prokaryotes)• DNA polymerase I-IV
• Polymerase III: Major DNA polymerase
• Polymerase I: Removes RNA primers
• Eukaryotes• DNA polymerase α, β, γ, δ, and ε
• Polymerase γ: located in mitochondria
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Primers
• DNA polymerase cannot initiate replication
• Primers: short nucleotide sequences
• Formed at point of initiation of new chain
• Required by DNA polymerase to function
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Primers
• DNA Primase: Makes primers
• Primers contain RNA• Ribonucleotides (not deoxy-ribonucleotides)
• Uracil instead of thymine
• Eventually removed and replaced with DNA
Ribonucleotide Deoxyribonucleotide
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Replication Fork
5’
3’
5’
3’
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DNA ReplicationDirectionality
A
C
T
G
Adenosine-TP
5’
3’
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DNA ReplicationDirectionality
A
C
T
G
5’
3’
DNAPolymerase
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DNA ReplicationDirectionality
• Always occurs in 5’ to 3’ direction
• Nucleotides added to 3’ end of growing strand
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Replication Fork
5’
3’
5’
3’
DNAPolymerase
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Replication Fork
5’
3’
5’
3’
DNAPolymerase
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Replication Fork
5’
5’
3’
3’
DNAPolymerase
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Replication Fork
5’
5’
3’
3’
DNAPolymerase
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Replication Fork
Masur/Wikipedia
Leading Strand
Lagging Strand
Okazaki fragments
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Primer Removal
• Okazaki fragments synthesized until primer reached
• RNA primer removed and replaced with DNA
• Prokaryotes: DNA polymerase I
• Eukaryotes: DNA polymerase delta
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DNA Ligase
• Joins Okazaki fragments
• Creates phosphodiester bonds
Wikipedia/Public Domain
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Topoisomerase
• Prevent DNA tangling
• Break DNA then reseal to relieve tension/twists
• Topoisomerase I• Break single strands of DNA then reseal
• Topoisomerase II• Break double strands then reseal
Topoisomerases
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TopoisomeraseClinical Correlations
• Quinolone antibiotics• Prokaryotic topoisomerases
• Chemotherapy agents• Eukaryotic toposiomerases
• Etoposide/teniposide
• Irinotecan, topotecan
• Anthracyclines
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DNA ReplicationKey Points
• Leading strand replication is continuous
• Lagging strand replication is discontinuous• Okazaki fragments
• DNA ligase
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DNA ReplicationKey Point
• Semi-conservative• New DNA: one old and one new strand
Adenosine/Wikipedia
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Proofreading
• DNA polymerase can correct errors
• Synthesizes in new strand 5’ to 3’ direction
• Wrong nucleotide added: Can move backwards• 3’ to 5’ direction
• Correct error
• Exonuclease activity: remove incorrect nucleotide
• DNA polymerase: “3’ to 5’ exonuclease activity”
• Significantly reduces error rate
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Replication Fork
5’
3’
5’
3’
DNAPolymerase
Thymidine
Adenosine
Guanosine
Cytosine
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Replication Fork
5’
3’
5’
3’
DNAPolymerase
Thymidine
Adenosine
Guanosine
Cytosine
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Replication Fork
5’
3’
5’
3’
DNAPolymerase
Thymidine
Adenosine
Guanosine
Cytosine
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Telomerase
• Telomeres: nucleotides at end of chromosomes
• Contain T-T-A-G-G-G sequences
• No place for RNA primer on lagging strand
• Major problem eukaryotic cells (non-circular DNA)
• Telomerase enzyme• Recognizes telomere sequences
• Adds these sequences to new DNA strands
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Telomerase
• Contains an RNA template
• Uses template to synthesize telomere DNA
• “RNA-dependent DNA polymerase”
• Similar to reverse transcriptase
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Uzbas, F/Wikipedia
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Telomerase
• Extends 3’ end of DNA
• Allows DNA polymerase to complete lagging strand
• Avoids loss of genes with duplication
Uzbas, F/Wikipedia
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Telomerase
• Found in hematopoietic stem cells• Allows controlled indefinite replication
• Other cells that divide indefinitely• Epidermis, hair follicles, intestinal mucosa
• Implicated in many cancers • Allows immortality
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DNA MutationsJason Ryan, MD, MPH
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Protein Synthesis
DNA
RNA
Proteins
Translation
Transcription
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Codons3 Nucleotide Sequences
DNA
RNA
Proteins
T A C
A U G
Methionine
Translation
Transcription
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Genetic Code
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DNA Mutations
• Errors in DNA
• Simple: One/few base(s) abnormal
• Complex: Gene deletions, translocations
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DNA Mutations
• Germ line mutations• DNA of sperm/eggs
• Transmitted to offspring
• Found in every cell in body
• Somatic mutations• Acquired during lifespan of cell
• Not transmitted to offspring
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Point Mutations
• Transition (more common): • Purine to purine A to G
• Pyrimidine to pyrimidine (C to T)
• Transversion: • Purine to pyrimidine (A to T)
• Pyrimidine to purine (C to G)
Cytosine
Thymine
Pyrimidines
Adenine Guanine
Purines
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Wobble
• Some transitions less likely to alter amino acids
• Genetic code: often same AA with altered base
UU-PyrimidineSame AA
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Silent Mutation
• Nucleotide substitution codes for same amino acid
• Often base change in 3rd position of codon
A – A – AU – U – U
PHE
A – A – GU – U – C
PHE
PhenylalaninePhenylalanine
DNARNA
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Nonsense Mutation
• Nucleotide substitution
• Result: Early stop codon• Nucleotide triplet
• Signals termination of translation of proteins
• UGA, UAA, UAG
A – C – CU – G – G
TRY
A – C – TU – G – A
DNARNA
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Missense Mutation
• Nucleotide substitution
• Result: Different amino acid
G – T – A – G – T – A –G – T – A – G – T – A C – A – U – C – A – U –C – A – U – C – A – U
HIS HIS HIS HIS
G – T – A – G – G – A –G – T – A – G – T – A C – A – U – C – C – U –C – A – U – C – A – U
HIS PRO HIS HIS
DNARNA
DNARNA
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Sickle Cell Anemia
• Root cause: Missense mutation beta globin gene
• Single base substitution 6th codon of β gene• Adenine changed with thymine
• Substitution of valine for glutamate in beta chains
G – A – GC – T – C G – A – G
GLU
G – T – GC – A – C G – U – G
VAL
ValineGlutamate
DNA
RNA
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Insertions and Deletions
• Addition/subtraction of nucleotides
• Can alter the protein product of a gene
• Cystic fibrosis• Most common mutation: delta F508
• Deletion of 3 DNA bases
• Loss of phenylalanine
• Abnormal protein folding
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Insertions and Deletions
• Addition/subtraction of nucleotides
• Can alter the protein product of a gene
• Cystic fibrosis• Most common mutation: delta F508
• Deletion of 3 DNA bases
• Loss of phenylalanine
• Abnormal protein folding
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Frameshift Mutation
• Insertion or deletion of nucleotides/bases
• Alters the reading frame
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G – T – A – G – T – A – G – T – A – G – T – A
HIS HIS HIS HIS
G – T – A – G – C – A – G – T – A – G – T – A
HIS ARG HIS HIS
Point Mutation
G – T – A – G – T – A – G – T – A – G – T – A
HIS HIS HIS HIS
A – G – T – A – G – T – A – G – T – A – G – T – A
SER SER SER
FrameshiftMutation
DNA Size Unchanged
DNA Size Changed
DNA
SER
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Frameshift Mutation
• Deletion/insertion not multiple of 3
• Misreading of nucleotides downstream
• Significant change to protein• Many amino acids may change
• Early stop codon → truncated protein
• Loss of stop codon → elongated protein
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Frameshift Mutation
• Described in Tay Sachs disease• Frameshift mutations (insertions/deletions)
• Gene for hexosaminidase A
• Duchenne muscular dystrophy• Dystrophin gene
• Frameshift deletions → absence of functional dystrophin
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Slipped-Strand MispairingDNA Slippage
• Occurs in areas of repeated nucleotide sequences
• Occurs with inadequate mismatch repair
• Insertions/deletions → frameshift mutations
A – A A – A
T – T T – T
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Slipped-Strand MispairingDNA Slippage
A – A
A – A
T – T T – T
A – A
T – T
Slippage in template strand → deletion (DNA not replicated)Slippage in replicated strand → insertion (replicated strand longer)
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Trinucleotide Repeat Disorders
• Occur in genes with repeat trinucleotide units• Example: CAGCAGCAGCAG
• Extra repeats in gene → disease
• Key examples• Fragile X syndrome
• Friedreich’s ataxia
• Huntington’s disease
• Myotonic dystrophy
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Microsatellite Instability
• Microsatellite• Short segments of DNA
• Repeated sequence (i.e. CACACACA)
• Mismatch repair enzyme failure → instability• Variation (instability) in size of segments among cells
• Seen in colon cancer
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DNA RepairJason Ryan, MD, MPH
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DNA Damage
• Occurs frequently in life of a cell• Heat, UV radiation, chemicals, free radicals
• Rarely leads to permanent damage
• Numerous repair enzymes/mechanisms exist
• Without repair, genetic material quickly lost
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Types of DNA Damage
• Depurination• Occurs spontaneously thousands of times per day
• Results in loss of purine bases (guanine and adenine)
• Deamination• Occurs spontaneously hundreds of times per day
• Base loses amine group (cytosine)
GuanineAdenine
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Types of DNA DamageDepurination
Guanosine
Guanine
Sugar Phosphate
AdenosineAdenine
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Types of DNA DamageDeamination
Cytidine
Cytosine Uracil
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Types of DNA Damage
• Free radicals or radiation damage base rings
• Oxidative damage, methylation, hydrolysis
Guanosine
Oxidative attack
Methylation
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Repair Mechanisms
• Single strand• Base excision
• Nucleotide excision
• Mismatch repair
• Double strand• Homologous end joining
• Non-homologous end joining
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Base Excision Repair
• Pathway for damaged DNA repair
• Recognize specific base errors• Deaminated bases, oxidized bases, open rings
• Numerous variations/enzymes used by cells
• Functions throughout the cell cycle (all phases)
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Base Excision Repair
• DNA glycosylases• Several different enzymes
• Remove damaged bases
• Creates a baseless nucleotide
• “Apurinic” or “apyrimidic” nucleotide
A T C G
T A CG
A C G
T A CG
GlycosidicBond
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Base Excision Repair
• AP endonuclease• Recognizes nucleotides without a base
• Attacks 5’ phosphate end of DNA strand
• “Nicks” damaged DNA upstream of AP site
• Create a 3'-OH end adjacent to the AP site
• AP lyase• Some DNA glycosylases also possess AP lyase activity
• Attack 3’ hydroxyl end of ribose sugar
A C G
T A CG
A C G
T A CG
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Base Excision Repair
Wikipedia/Public Domain
APEndonuclease
APLyase
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Base Excision Repair
• DNA polymerase• Adds new nucleotide (complementary to opposite base)
• Extends 3'-OH terminus
• DNA ligase seals strand
A C G
T A CG
A C G
T A CG
T
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Nucleotide Excision Repair
• Removes “bulky” DNA damage• Multiple bases
• Often pyrimidine dimers
• Commonly caused by UV radiation (sunlight)
• G1 phase (prior to DNA synthesis)
• Endonucleases removed multiple nucleotides
• DNA polymerase and ligase fill gap
Public Domain
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Nucleotide Excision Repair
Thymidine Thymidine
CyclobutanePyrimidine
Dimer
Wikipedia/Public Domain
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Xeroderma Pigmentosum
• Defective nucleotide excision repair
• Extreme sensitivity to UV rays from sunlight
• Signs appear in infancy or early childhood
• Very easy sunburning
• Freckling of skin
• Dry skin (xeroderma)
• Changes in skin pigmentation
• Very high risk of skin cancer• May develop in childhood
James Halpern, Bryan Hopping and Joshua M Brostoff
![Page 76: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/76.jpg)
Mismatch Repair
• Identifies incorrectly placed bases/nucleotides• Insertions, deletions, incorrect matches
• Occurs when proofreading misses errors
• No damage to base – not recognized by repair systems
• Occurs in S/G2 phase (after DNA synthesis)
• Newly synthesized strand compared to template
• Nucleotide errors removed and resealed
![Page 77: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/77.jpg)
Mismatch Repair
• Important for microsatellite stability
• DNA has many repeating segments
• “Microsatellites”
AC
AAACCCCCCAAA
GGTTTTGG
![Page 78: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/78.jpg)
Mismatch Repair
• DNA slippage can occur at repeats
• Results in a mismatch
• Repaired by MMR systems
• Result: number of repeats (microsatellites) stable
![Page 79: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/79.jpg)
Mismatch Repair
• Microsatellite instability• Results if MMR systems deficient
• Seen in cancers cells (colon cancer)
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HNPCCHereditary Non-Polyposis Colorectal Cancer/Lynch Syndrome
• Germline mutation of DNA mismatch repair enzymes• About 90% due to MLH1 and MSH2 mutations
• Leads to colon cancer via microsatellite instability• About 80% lifetime risk
• Hallmark: cancer cells with microsatellite instability
![Page 81: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/81.jpg)
Double Strand Damage
• Commonly result from exogenous sources • Ionizing radiation
• Caused by radiation therapy (cancer)
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Homologous End Joining
• Homology = similar structure
• HEJ = uses sister chromosome template
SisterChromosome
Template
![Page 83: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/83.jpg)
Non-Homologous End Joining
• Uses many proteins to re-join broken ends• DNA pol λ and μ re-extend the ends
• Many other enzymes
• No template used (non-homologous)
• Highly error-prone
Double Strand Break(ionizing radiation)
NHEJ
![Page 84: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/84.jpg)
Fanconi Anemia
• Inherited aplastic anemia
• More than 13 genetic abnormalities identified
• Many involve DNA repair enzymes• Hypersensitivity to DNA damage
• Cells vulnerable to DNA strand cross-links
• Also impaired homologous recombination
![Page 85: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/85.jpg)
Ataxia Telangiectasia
• Defective Nonhomologous end-joining (NHEJ)• Mutations in ATM gene on chromosome 11
• Ataxia Telangiectasia Mutated gene
• Repairs double stranded DNA breaks via NHEJ
• DNA hypersensitive to ionizing radiation
• CNS, skin, immune system affected
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Ataxia TelangiectasiaClinical Features
• Most children healthy for first year
• Begin walking at normal age but slow development
• Progressive motor coordination problems
• By 10 years old, most in wheelchairs
• Other symptoms• Recurrent sinus/respiratory infections (immune system)
• Telangiectasias (skin)
• High risk of cancer
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TranscriptionJason Ryan, MD, MPH
![Page 88: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/88.jpg)
Transcription
• Synthesis of RNA• Ribonucleotides (not deoxyribonucleotides)
• Uridine (not thymidine)
• DNA used as template
Thymidine Uridine
![Page 89: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/89.jpg)
Transcription
Thymidine
CytidineAdenosine
Guanosine
3’
3’
5’
5’
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Transcription
Thymidine
CytidineAdenosine
Guanosine
3’
5’Template Strand
3’
5’
RNA Synthesis 5’ to 3’
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Transcription
Thymidine
CytidineAdenosine
Guanosine
3’
5’
3’
Uridine
Template Strand3’
5’
5’
Coding Strand
![Page 92: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/92.jpg)
Transcription
GTCA
GUCA
DNA
RNA
Transcription
![Page 93: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/93.jpg)
Types of RNA
• Messenger RNA• Longest chains of RNA
• Nucleotides specify amino acids
• Used the synthesize proteins
• Ribosomal RNA• Form ribosomes
• Transfer RNA• Transfer amino acids to proteins
![Page 94: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/94.jpg)
Types of RNA
• Micro RNA (miRNA)• Regulate gene expression
• Target mRNA molecules → bind via base pairing
• Block translation into protein
• Small interfering RNA (siRNA)• Also regulate gene expression
• Cause degradation of mRNA
• Small nuclear RNA (snRNA)• Splicing of pre-mRNA
![Page 95: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/95.jpg)
RNA Polymerase
• Synthesizes RNA from DNA template
• Does not require a primer (like DNA polymerase)
• Binds promoter regions of DNA
• Requires transcription factors (proteins)
• Binds DNA → opens double helix
![Page 96: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/96.jpg)
RNA Polymerase
• Prokaryotes: One RNA polymerase• Multi-subunit complex
• Makes all types of RNA
• Eukaryotes: multiple RNA polymerase enzymes
• RNA polymerase I: most rRNA (5.8S, 18S, 28S)
• RNA polymerase II: mRNA
• RNA polymerase III: rRNA (5S), other RNAs
![Page 97: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/97.jpg)
RNA Polymerase Inhibitors
• Alpha amanitin• Protein found in Amanita phalloides (death cap mushrooms)
• Powerful inhibitor of RNA polymerase II
• Liver failure (taken up by liver cells)
Newnam/Wikipedia
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RNA Polymerase Inhibitors
• Rifampin• Inhibits bacterial RNA polymerase
• Used to treat tuberculosis
• Actinomycin D • Used as chemotherapy
• Inhibits RNA polymerase
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Transcription Factors
• Additional proteins required to initiate transcription
• Prokaryotes• Protein factor (σ factor)
• Eukaryotes• Multiple factors (“transcription factors”)
• Many bind RNA polymerase II
• TFIID, TFIIB, TFIIE, etc
![Page 100: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/100.jpg)
Promoters
• DNA regions
• Not transcribed
• Bind RNA polymerase and transcription factors
• Bound RNA polymerase opens double helix
Promoter Gene
Transcription StartPoint
3’5’
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Promoters
• TATA Box• Very common eukaryotic promoter
• TATAAA
• Binds transcription factors (TFIID)
• CAAT Box• CCAAT sequence
• GC Box• GGGCGG
![Page 102: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/102.jpg)
Enhancers
• DNA sequences that increase rate of transcription
• May be upstream or downstream of gene they regulate
• Binds transcription factors called activators
• Because of DNA coiling, many are geometrically close but many nucleotides away from gene
• Stabilize transcription factors/RNA polymerase
![Page 103: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/103.jpg)
Silencers
• DNA sequence that decreases rate of transcription
• May be upstream or downstream of gene they regulate
• Binds transcription factors called repressors
• Repressors prevent RNA polymerase binding
![Page 104: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/104.jpg)
Untranslated Regions
• Portions of mRNA at 5’ and 3’ ends
• Not translated into protein
• 5’ UTR upstream from coding sequence• Recognized by ribosomes to initiate translation
• 3' UTR found following a stop codon• Important for post-transcriptional gene expression
mRNA UTRUTR5’ 3’
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Protein SynthesisProkaryotes
DNA
mRNA
Proteins
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Protein SynthesisEukaryotes
I
I
Proteins
E I E
E I E
E E
E ECytoplasm
Nucleus
Introns = stay IN nucleusExons = exit nucleus
DNA
RNA
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mRNA in Eukaryotes
• Initial transcript: hnRNA• Heterogeneous nuclear RNA
• Also called pre-mRNA
• hnRNA modified to become mRNA
• Three key modifications before leaving nucleus• 5’ capping
• Splicing out of introns
• 3’ polyadenylation
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5’ Capping
• Addition of 7-methylguanosine to 5’ end
• Added soon after transcription begins
• Distinguishes mRNA from other RNA
Zephyris/Wikipedia
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RNA Splicing
• Occurs during transcription
• Introns removed from mRNA in nucleus
• Introns always have two nucleotides at either end
• 5' splice site: GU
• 3' splice site: AG
Exon GU ExonAG3’5’
mRNA
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RNA Splicing
• Primary transcript combines with snRNPs• Small nuclear ribonucleoproteins (snRNPs)
• Short RNA polymers complexed with proteins
• RNAs contain high content of uridine (U-RNAs)
• Five different U-RNAs defined: U1, U2, U4, U5, and U6
![Page 111: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/111.jpg)
RNA Splicing
• snRNPs and mRNA forms “spliceosome”
• Loop of mRNA with intron is formed (“lariat”)
• Lariat released → removes intron
• Exons joined
![Page 112: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/112.jpg)
RNA Splicing
BCSteve/Wikipedia
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Antibodies
• Anti-Sm (anti-smith)• Antibodies against proteins in snRNPs
• Seen in patients with SLE
• Anti-RNP• Antibodies against proteins associated with U1 RNA
• Strongly associated with Mixed Connective Tissue Disease
• Also seen in SLE, Scleroderma
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Alternative Splicing
• Allows many proteins from same gene
• DNA: Exon1 – Exon 2 – Exon 3 – Exon 4 … Exon 10
• Protein 1: Exon1 – Exon 3 – Exon 7
• Protein 2: Exon 2 – Exon 5 - Exon 10
![Page 115: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/115.jpg)
Alternative Splicing
Wikipedia/Public Domain
![Page 116: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/116.jpg)
Splicing Errors
• Can lead to disease• Loss of exons, retention of introns
• Incorrect joining of introns
• Beta thalassemia• Many mutations described
• Some involve splice sites
• Oncogenesis• Many splice site mutations/errors described
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3’ Polyadenylation
• Occurs at termination of mRNA transcription
• Triggered by specific DNA/RNA sequences
• “Polyadenylation signal:” AAUAAA
• AAUAAA followed by 10-30 nucleotides then CA
AATAAA CA
AAUAAA CA
DNA(coding strand)
mRNA
3’5’
5’ 3’
![Page 118: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/118.jpg)
3’ Polyadenylation
• Requires several RNA binding proteins
• Cleavage and polyadenylation specificity factor (CSF)• Binds AAUAAA
• Cleavage stimulation factor (CstF)• Binds CA sequence
• Leads to termination of DNA transcription
AAUAAA CAmRNA
3’
![Page 119: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/119.jpg)
3’ Polyadenylation
• Enzyme: Poly-A polymerase (PAP)
• Adds ~200 adenosine nucleotides to 3’ end mRNA
• No template
AAAAAA
PAP
AAUAAA CAmRNA
3’
AAUAAAmRNA
3’
![Page 120: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/120.jpg)
TranscriptionSummary
CAAT TATA Exon Intr Exon Intr AATAA
TranscriptionStart
Promoter
Exon Exon AAUAA
DNA Coding Strand
mRNA
3’5’
3’5’
Cap AAAA
Cytoplasm
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MicroRNAmiRNA
• Important regulatory molecules for mRNA
• Regulate mRNA expression to proteins
• Bind mRNA via base pairing
• Extensive binding can remove poly-A tail
• Exposes mRNA to degradation by endonucleases
• Modifies gene expression at mRNA level
![Page 122: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/122.jpg)
Processing BodiesP-bodies
• Some mRNA moved to P-bodies in cytoplasm• Seen with less extensive miRNA binding
• mRNA sequestered from ribosomes
• Often degraded
• Some evidence that mRNA may later be translated
![Page 123: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/123.jpg)
TranslationJason Ryan, MD, MPH
![Page 124: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/124.jpg)
TranscriptionSummary
CAAT TATA Exon Intr Exon Intr AATAA
TranscriptionStart
Promoter
Exon Exon AAUAA
DNA Coding Strand
mRNA
3’5’
3’5’
Cap AAAA
Cytoplasm
mRNA read 5’ to 3’
![Page 125: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/125.jpg)
Translation
• Synthesis of protein using mRNA as template
• Occurs in cytoplasm on ribosomes
• tRNA brings amino acids to ribosome for assembly
![Page 126: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/126.jpg)
Translation
Boumphreyfr /Wikipedia
![Page 127: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/127.jpg)
Ribosomes
• Some are “free” in cytoplasm
• Also bound to the endoplasmic reticulum • Forms rough ER
• Contain rRNA and proteins
• Arranged as a large and small subunit
• Size measured in Svedberg units• Measure of rate of sedimentation by centrifugation
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Ribosomes
• Prokaryotes • 70S ribosomes
• Small (30S) and large (50S) subunit
• Small subunit: 16S RNA plus proteins
• Large subunit: 5S RNA, 23S RNA, plus proteins
• Protein synthesis inhibitor antibiotics• Aminoglycosides, others
• Target components of bacterial ribosomes
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Ribosomes
• Eukaryotes• 80S ribosomes
• Small (40S) and large (60S) subunits
• Small subunit: 18S RNA plus proteins
• Large subunit: 5S RNA, 28S RNA, 5.8S RNA plus proteins
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tRNA
• Transfers amino acids to protein chains
• Synthesized by RNA polymerase III
• Many bases are chemically modified
N,N dimethyl Guanosine Guanosine
![Page 131: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/131.jpg)
tRNA
• Cloverleaf shape (secondary structure)
• Base pairing within molecule
• 70-90 nucleotides in length (tiny)
• Key portions• Anticodon
• D loop (part of D arm)
• T loop (part of T arm)
• 3’ end
Yikrazuul
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Anticodon
• 3 nucleotides on tRNA
• Pairs with complementary mRNA
• Correct pairing → correct protein synthesis
Boumphreyfr /Wikipedia
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Genetic Code
![Page 134: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/134.jpg)
D loop
• Contains dihydrouridine
• tRNA recognition by aminoacyl-tRNA synthetase
Uridine
Dihydrouridine
![Page 135: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/135.jpg)
T loop
• Contains a TΨC sequence• T = Ribothymidine
• Ψ = Pseudouridine
• C = Cytidine
• Needed for tRNA ribosome binding
PseudouridineRibothymidineUridine
![Page 136: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/136.jpg)
3’ End
• Always ends in CCA
• Hydroyxl (OH) of A attaches to amino acid
Yikrazuul
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Charging
• Process of linking amino acids to tRNA
• Each tRNA linked to one amino acid
• Catalyzed by Aminoacyl-tRNA synthetase
• Adds amino acid to tRNA
• Requires ATP
![Page 138: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/138.jpg)
tRNA
ATP
AMP
Amino AcidAMP
ChargedtRNA
AminoacylGroup
![Page 139: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/139.jpg)
Aminoacyl-tRNA synthetase
• One enzyme per amino acid in most eukaryotic cells• i.e. one enzyme attaches glycine to correct tRNA;
Dancojocari/Wikipedia
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tRNA
• Many amino acids have similar structures
• Mischarged tRNA→wrong AA for mRNA codon
• Hydrolytic editing• Aminoacyl-tRNA synthetase scrutinizes amino acid
• If incorrect → hydrolyzes from AMP or tRNA
• Increases accuracy of charging tRNA
![Page 141: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/141.jpg)
Protein Synthesis
• Amino acids: N-terminal and C-terminal ends
• Proteins synthesis: addition to C-terminal end
![Page 142: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/142.jpg)
Protein Synthesis
• Three stages:• Initiation
• Elongation
• Termination
![Page 143: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/143.jpg)
Protein Synthesis
• Ribosomes: Four binding sites• One for mRNA
• Three for tRNA: A-site, P-site, E-site
3’5’P AE
![Page 144: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/144.jpg)
Protein Synthesis
• A-site: Amino acid binding (charged tRNA)
• P-site: tRNA attached to growing protein chain
• E-site: Exit of tRNA
3’5’P AE
![Page 145: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/145.jpg)
Initiation
• Begins with AUG on mRNA
• Codes for methionine or N-formylmethionine (fMet)
• Binds directly to P-site
• Usually removed later by protease enzymes
• fMET = chemotaxis of neutrophils (innate immunity)
Methionine N-formylmethionine
![Page 146: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/146.jpg)
Initiation
• Uses GTP hydrolysis
• In eukaryotes require initiation factors (proteins)• Assemble ribosomes and tRNA
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Elongation
• Usually divided into a sequence of four steps
• Uses elongation factors (proteins)• Bacteria: EF-Tu and EF-G
• Eukaryotes: EF1 and EF2
• Hydrolyze GTP to GDP
• EF2: Target of bacterial toxins• Diphtheria toxin (Corynebacterium diphtheriae)
• Exotoxin A (Pseudomonas aeruginosa)
• Inhibits protein synthesis
![Page 148: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/148.jpg)
Protein Synthesis
• Step 1: Charged tRNA binds A-site• P-site and A-site next to one another
t
3’5’
NH2
t
P AE
![Page 149: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/149.jpg)
Protein Synthesis
• Step 2: Amino acid joined to peptide chain• Catalyzed by ribosome (“ribozyme”)
• Peptidyl transferase: Part of large ribosome (made of RNA)
• Protein attached to A-site
tt
P A3’5’
NH2
E
![Page 150: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/150.jpg)
Protein Synthesis
• Step 3: Ribosome moves down mRNA toward 3’ end• “Translocation”
• Protein moves to P-site
tt
P A3’5’
NH2
E
![Page 151: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/151.jpg)
Protein Synthesis
• Step 4: tRNA leaves E-site
tt
P A3’5’
NH2
E
![Page 152: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/152.jpg)
Termination
• Translation ends at mRNA stop codons• UAA, UAG, UGA
• Not recognized by tRNA
• Do not specific an amino acid
• Releasing factors bind to ribosome at stop codons
• Catalyze water added to protein chain
NH2
OH
![Page 153: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/153.jpg)
Posttranslational Modifications
• Creates functional protein
• Folding
• Addition of other molecules
![Page 154: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/154.jpg)
Posttranslational Modifications
• Phosphorylation• Amino acid residue phosphorylated
• Protein kinase enzymes add phosphate group
• Glycosylation• Formation of the sugar–amino acid linkage
• Many linkages: N-, O-, C-linked glycosylation
• Creates glycoproteins
![Page 155: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/155.jpg)
Posttranslational Modifications
• Hydroxylation• Addition of hydroxyl (OH) groups
• Important for collagen synthesis
• Hydroxylation of proline and lysine residues
ProlineHydroxyproline
![Page 156: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/156.jpg)
Posttranslational Modifications
• Methylation• Addition of methyl (CH3) groups
• Acetylation• Addition of acetyl (CH3CO) group
• Ubiquitination• Addition of ubiquitin (small protein)
• Tags proteins for destruction in proteasome
Acetyl Group
![Page 157: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/157.jpg)
Chaperones
• Proteins that facilitate folding
• Bind to other proteins → ensure proper folding
• Classic example: Heat shock proteins• Family of proteins
• Also called stress proteins
• Constitutively expressed
• Increased expression with heat, pH shift, hypoxia
• Stabilize proteins; maintain protein structure
• Help cells survive environmental stress
![Page 158: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/158.jpg)
Polymerase Chain ReactionJason Ryan, MD, MPH
![Page 159: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/159.jpg)
PCRPolymerase Chain Reaction
• Laboratory technique
• Amplifies (copies) DNA molecules in a sample
• Uses:• Make more DNA from small amount
• Determine if DNA is present (i.e. does it amplify?)
• Determine amount of DNA (i.e. how quickly does it amplify?)
PCR
![Page 160: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/160.jpg)
PCRIngredients
• Sample (DNA)
• DNA polymerase
• Primer• Single-stranded DNA segment
• Complementary to DNA under evaluation
• Nucleotides
GTCA
Primer
![Page 161: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/161.jpg)
PCRTechnique
• Heat sample• DNA denatures into single strands
• Cool sample• Primer anneals (binds) complementary DNA (if present)
• Warm sample• DNA polymerase elongates from primer
• Process repeated in cycles
• Each cycle generates more DNA
![Page 162: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/162.jpg)
PCR
Enzoklop/Wikipedia
![Page 163: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/163.jpg)
Real Time PCRQuantitative PCR
• PCR done in presence of fluorescent dye
• Amount of dye proportional to amount of DNA
• More DNA = more fluorescence
• Fluorescence detected as PCR ongoing
• Rapid increase florescence = more DNA in sample
![Page 164: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/164.jpg)
PCRUses
• Herpes simplex virus encephalitis• DNA in CSF
• HIV Viral Load• Uses reverse transcriptase to make cDNA
• Amplification of cDNA
• Amount of cDNA produced over time indicates viral load
• Standard tool for monitoring viral load
![Page 165: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/165.jpg)
BlottingJason Ryan, MD, MPH
![Page 166: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/166.jpg)
Blotting
• Laboratory techniques
• Southern blot: Identifies DNA
• Northern blot: Identifies RNA
• Western blot: Identifies proteins
![Page 167: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/167.jpg)
Southern Blot
• Named for inventor (Edward Southern)
• Uses a probe to identify presence of DNA in a sample
![Page 168: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/168.jpg)
Probe
• Single-stranded DNA molecule
• Carries radioactive or chemical markers
• Binds complementary sequences• Probe called “cDNA”
• “Hybridization”
• Once bound, markers reveal DNA in sample
3’5’
DNA in Sample
Probe
![Page 169: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/169.jpg)
Southern BlotStep 1
DNA Sample
Restriction nucleases(enzymatically cleavage)
![Page 170: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/170.jpg)
Southern BlotStep 2
Gel ElectrophoresisSize separation
![Page 171: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/171.jpg)
Gel Electrophoresis
Jeffrey M. Vinocur
![Page 172: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/172.jpg)
Southern BlotStep 3
BlottingTransfer to filter paper
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Southern BlotStep 4
• Add probe
• Wash away unbound probe
• Only bound probe remains
• Filter paper exposed to film → bound DNA revealed
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Southern BlotStep 4
• Often done with multiple samples
Sample 1 Sample 2
![Page 175: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/175.jpg)
Southern BlotDNA
Electrophoresis
DNA probe
![Page 176: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/176.jpg)
Southern BlotClinical Uses
• Restriction fragment length polymorphisms
• Sickle cell anemia
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RFLPRestriction fragment length polymorphisms
• Restriction nucleases• DNA cutting enzymes
• Cut DNA at specific base sequences (i.e. GTGCAC)
• Restriction fragment length polymorphisms• Analysis of fragments of DNA from restriction nucleases
• Different genes = different length of fragments
• Southern blotting to detect lengths after fragmentation
1.5kb
1.0kb
1.3kb
Gene A Gene B
![Page 178: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/178.jpg)
RFLPRestriction fragment length polymorphisms
Wikipedia/Public Domain
![Page 179: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/179.jpg)
Sickle Cell Anemia
• Normal β-globin gene: Two fragments• 1.15kb and 0.2kb
• Sickle cell: One fragment • 1.35kb
• This fragment seen only with HbS gene
1.15kb
1.35kb
Normal Carrier SS
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Northern BlotRNA
Electrophoresis
Probe
Useful for assessingmRNA levels
(gene expression)
![Page 181: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/181.jpg)
Western BlotProtein
Electrophoresis
Antibody
![Page 182: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/182.jpg)
Western Blot
• Detection of antibodies• IgG or IgM in Lyme disease
• IgG HIV-1
![Page 183: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/183.jpg)
Southwestern Blot
• Used to study DNA-protein interaction
• Combines features of Southern and Western blots
• Proteins separated by electrophoresis (Western)
• DNA probe added (Southern)
• Used for studying DNA-binding proteins
• Especially transcription factors
![Page 184: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/184.jpg)
Southwestern BlotProtein
Electrophoresis
DNA
![Page 185: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/185.jpg)
Flow CytometryJason Ryan, MD, MPH
![Page 186: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/186.jpg)
Flow Cytometry
• Flow = motion of fluid
• Cytometry = measurement of cells
• Flow cytometry = Analysis of cells as they flow in a liquid through a narrow steam
• Key point: Used to analyze cells• By size
• By surface proteins
![Page 187: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/187.jpg)
Flow Cytometer
• Key components:• Flow cell: moves cells through machine
• Laser: light scattered by cells
• Photodetector: detects light scatter
Biol/Wikipedia
![Page 188: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/188.jpg)
Flow Cytometer
Photodetector
Light Source
![Page 189: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/189.jpg)
Flow Cytometer
Light Forward ScatterSize
Side ScatterGranularity
![Page 190: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/190.jpg)
Flow Cytometry
Front Scatter
Sid
e Sc
atte
r
Lymphocytes
Monocytes
Granulocytes
![Page 191: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/191.jpg)
Antibody Staining
• Specific antibodies to surface/intracellular proteins
• Tagged with unique fluorochrome
• Flow cytometer detects fluorochrome
• Indicates presence of protein in cells
![Page 192: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/192.jpg)
Antibody Staining
CD8
CD
4
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Flow CytometryClinical Uses
• Fetal maternal hemorrhage• Fetal red cells cross placenta to maternal blood
• Seen with placental failure/trauma
• Presents as decreased fetal movement, abnormal fetal HR
• Can cause stillbirth
• Flow cytometry: monoclonal antibody to hemoglobin F
• Detects fetal hemoglobin in red cells
Øyvind Holmstad/Wikipedia
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Flow CytometryClinical Uses
• Paroxysmal nocturnal hemoglobinuria• Fluorescently-labeled monoclonal antibodies
• Bind glycosylphosphatidylinositol (GPI) anchored proteins
• Decay Accelerating Factor (DAF/CD55)
• MAC inhibitory protein (CD59)
• Reduced or absent on red blood cells in PNH
Databese Center for Life Science (DBCLS)
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ELISAJason Ryan, MD, MPH
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ELISAEnzyme-linked immunosorbent assay
• Detects antigens and antibodies in serum
• Based on enzymatic color change reaction
• Several forms• Direct
• Indirect
• Sandwich
• Competitive
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ELISAEnzyme-linked immunosorbent assay
Jeffrey M. Vinocur
![Page 198: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/198.jpg)
Direct ELISA
• Add serum to be tested
• Serum coats plate → antigen secured to surface
• Wash away fluid
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Direct ELISA
• Add enzyme-labeled antibody specific to antigen
• Wash away unbound antibodies
• Add substrate → color change
• Enzyme-linked antibodies directly bind antigen
E E E
S S S
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Indirect ELISA
• Add serum for analysis (like direct)
• Add antibody to antigen of interest
• Antibody not enzyme linked
• Wash away unbound antibody
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Indirect ELISA
• Add enzyme-labeled secondary antibody
• Substrate → color change → identification of antigen
• Result: Identifies presence of antigen in serum
• Enzyme-linked antibodies indirectly bind antigen
E E E
S S S
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ELISADirect vs. Indirect
• Direct• Fewer steps
• Specific antibody must be enzyme-linked
• Time-consuming to label antibodies to unique antigens
• Indirect• More steps
• Specific antibody NOT enzyme-linked
• Specific antibody easier to acquire (i.e. mouse antibody)
• Secondary antibody easier to acquire (i.e. anti-mouse IgG)
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Sandwich ELISA
• Plate coated with capture antibody
• Sample added → any antigen present binds
• Detecting antibody added → binds to antigen• Direct: detecting antibody enzyme linked
• Indirect: secondary enzyme-linked antibody added
• Substrate added → color change
E E E
S S S
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Sandwich ELISA
• High specificity• Two antibodies used
• Unlikely to bind wrong antigen
• Works with complex samples• Antigen does not require purification
• Can use secondary antibody like indirect
![Page 205: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/205.jpg)
Competitive ELISA
• Primary antibody incubated with sample
• Antigen-antibody complexes form
• More antigen = more binding = less free antibody
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Competitive ELISA
• Mixture added to antigen coated plates
• Unbound antibody binds antigen
• Wash away antigen-antibody complexes
• Secondary antibody and substrate added
• More color change = LESS antigen in sample
![Page 207: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/207.jpg)
Competitive ELISALots
of antigen Less color
Lessantigen MORE
color
Mixture added to
plates
Wash away
Enzymeadded
![Page 208: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/208.jpg)
ELISA Uses
• HIV antibody detection• Indirect method (many variants used)
• HIV antigen attached to well
• Sample reacts with antigen-coated plate
• 2° antibody: antihuman immunoglobulin with bound enzyme
• Addition of substrate results in color change
E E E
S S S
![Page 209: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/209.jpg)
ELISA Uses
• HIV p24 antigen detection• Often sandwich ELISA used (many variants)
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Microarraysand FISHJason Ryan, MD, MPH
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DNA Microarray
• Also called DNA chip or biochip
• Solid structure: glass, plastic, or silica
• Thousands of DNA sequences (probes) attached
• Used to test a sample DNA with fluorescent markers
• Sample hybridizes with complementary bases
• Computer detects which probes bind sample
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DNA Microarray
Wikipedia/Public Domain
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DNA Microarray
Schutz/Wikipedia
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DNA Microarray
• Gene expression• Which genes active/inactive
• Example: cancer cells versus normal cells
• Cellular mRNA collected → cDNA
• cDNA tested using microarray
• Determines gene expression
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DNA Microarray
Wikipedia/Public Domain
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DNA Microarray
• Copy number variation• Some cells contain ↓/↑ copies of genes/DNA
• Increased/decreased copies linked to disease
• Cellular DNA collected →microarray testing
• Reference sample also tested
• Results (fluorescence intensities) compared• Sample = reference (no extra copies)
• Sample > reference (more copies)
• Sample < reference (fewer copies)
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DNA Microarray
• Single nucleotide polymorphisms (SNPs)• Genes exist with variations in a single nucleotide
• Variations represented in the microarray
• Cellular DNA collected → tested using microarray
• Binding indicates which SNP present in sample gene
• Many SNPs associated with disease
• Many SNPs preserved within families
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FISHFluorescence in situ hybridization
• Fluorescent DNA probe binds to specific gene site
• Localizes genes to a chromosome
• Determine which chromosome contains gene
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FluorescentDNA Probe
DNA ofInterest Hybridization
FISHFluorescence in situ hybridization
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FISHFluorescence in situ hybridization
• Often done on cells in metaphase • Cells arrested in mitosis
• Chromosomes visible individually
• Fixed to glass slide
• DNA probes used that match regions of known chromosomes
• Probes hybridized to chromosomes on slide
• “in situ” hybridization
• Probes visualized with fluorescence microscopy
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FISHFluorescence in situ hybridization
• Often used to compare test cell to normal cells• Locate gene in test cells
• Microdeletion: no fluorescence of chromosome • 22q11(DiGeorge syndrome)
• Translocation: fluorescence on different chromosome
• Duplication: extra site of fluorescence
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Cell CycleJason Ryan, MD, MPH
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Cell Cycle
Richard Wheeler (Zephyris) 2006
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Cell Cycle
Richard Wheeler (Zephyris) 2006
Interphase(Growth)
M phase(Mitosis)
G1 (growth)S (synthesis)G2 (growth)
G0(resting)
![Page 225: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/225.jpg)
Cell Cycle
• G1 phase• Synthesis of proteins, organelles
• Length varies depending on conditions
• Mitogens: • Extracellular signaling molecules, usually proteins
• Stimulate cell division
• Function via cyclin dependent kinases (Cdks)
• Growth factor: Stimulates growth in size
• Some molecules both mitogens and GFs
• Terms sometimes used interchangably
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Cell Cycle
• S phase• Synthesis of DNA
• Chromosomes → two sister chromatids
• G2 phase• Growth in preparation for mitosis
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G0 Phase
• May occur in absence of mitogen stimulation
• Specialized non-dividing state
• Most cells in our body are in G0
• Some permanent G0
• Others go in/out
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G0 Phase
• Neurons, skeletal muscle cells• Permanent G0 state (“terminally differentiated”)
• Liver cells• Often in G0 but may divide if stimulated
• Fibroblasts, lymphocytes• Enter and exit G0 many times in their lifespan
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G0 Phase
• Bone marrow cells, GI epithelial cells, hair follicles• “Labile cells”
• Rapidly dividing
• Rarely/never enter G0
• Most effected by many forms of chemotherapy
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Mitosis
• Shortest (most rapid) portion of cell cycle
• Divided into phases• Prophase
• Prometaphase
• Metaphase
• Anaphase
• Telophase
Richard Wheeler (Zephyris) 2006
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MitosisProphase
• Chromosomes condense
• Spindle fibers forms
Ali Zifan/Wikipedia
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MitosisPrometaphase
• Chromosomes organize on mitotic spindle
Ali Zifan/Wikipedia
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MitosisMetaphase
• Chromosomes line up on metaphase plate
Ali Zifan/Wikipedia
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MitosisAnaphase
• Chromosomes separate
Ali Zifan/Wikipedia
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MitosisTelophase/Cytokinesis
• Spindle breaks down
• Cell divides
Ali Zifan/Wikipedia
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Cell Cycle Control
• Cells regulate progression through “checkpoints”• Also called “restriction points”
• G1-S (prior to S phase entry)
• G2-M (prior to mitosis)
• M phase (prior to anaphase/cytokinesis)
• Arrests cell if conditions not appropriate
• First checkpoint: Late G1 (G1-S)• Cell commits to cell cycle/growth
Richard Wheeler (Zephyris) 2006
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Cell Cycle Control
• Cyclin Dependent Kinases (Cdks)• Central components of cell cycle control
• Kinase enzymes (lead to phosphorylation of other proteins)
• Always present in cells but inactive
• Depend on cyclins to activate
• Cyclins: regulatory proteins – activate Cdks
• Cyclin-Cdk complexes• Phosphorylate regulatory proteins
• Allow progression through cell cycle
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Cyclins
• Many classes/subtypes
• Levels vary during cell cycle
Wikipedia/Public Domain
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G1-S Checkpoint
• During G1 phase → Cdk activity suppressed
• Mitogens activate Cdk→ entry into S phase• Interact with cell surface receptors
• Activate intracellular pathways
• Increase G1 cyclin levels
• Increase Cdk activity
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G1-S Checkpoint
• Cyclin-Cdk complexes activate E2F proteins• Transcription factors
• Bind to DNA promoter regions
• Activate genes for S phase
• E2F normally inhibited • Inhibited by E2F binding to retinoblastoma proteins (Rb)
• Inhibition released by G1-S-Cdk phosphorylation of Rb
• Rb regulates cell growth• “Tumor suppressor”
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G1-S Checkpoint
E2F
Active
Inactive
Cdks
E2F
PP
P
Cell Growth
Cyclin
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G1-S Checkpoint
• DNA damage can arrest cell division • Allows for repair
• Prevents development of mutant cells/cancer
• DNA damage initiates signaling pathways
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G1-S Checkpoint
• ATM pathway: Activated by double strand breaks• ATM: Ataxia Telangiectasia Mutated
• ATM gene mutation → Ataxia Telangiectasia
• ATR pathway: Single stranded breaks
• Both lead to phosphorylation of proteins
• Causes cell cycle/growth arrest
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P53 Protein
• Major target of ATM/ATR systems
• Phosphorylated after DNA damage• Prevents p53 breakdown
• Increases levels/activity
• p53 induces transcription of p21 protein
• p21 binds to Cdks→ inhibits Cdk activity
• Blocks cell progression through cell cycle
• p53/p21 = tumor suppressors
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P53 Protein
P53Unstable Protein
Rapid Breakdown
P53
P21 gene
DNA Damage
P53
StableP21
Cdk InhibitionGrowth Arrest
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G1-S Checkpoint
E2F
Active
Inactive
Cdks
E2F
PP
P
Cell Growth
Cyclin
P21
X
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G1-S Checkpoint
Richard Wheeler (Zephyris) 2006
Stimulation (Rb-E2F)DNA Damage (p53)
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Retinoblastoma
• Rare childhood eye malignancy
• Mutations in RB1 gene • Codes for Rb protein
• Abnormal Rb→ Unregulated cell growth (via E2F)
• Inherited forms: child born one mutated Rb gene• Initially child is heterozygous
• Loss of heterozygosity → cancer
Wikipedia/Public Domain
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Li-Fraumeni Syndrome
• Syndrome of multiple malignancies at an early age • Sarcoma, Breast, Leukemia, Adrenal Gland
• “SBLA” cancer syndrome
• Mutation in tumor suppressor gene TP53
• Codes for p53 protein
• Mutation: Cycle not arrested to allow for DNA repair
• Cells fail to undergo apoptosis
• Accumulation of damage →malignancy
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Human Papilloma Virus
• Associated with cervical cancer
• Expresses HPV oncogene E6
• Leads to synthesis of E6 protein
• Binds p53 protein → p53 degradation
• Degradation via ubiquitination• p53 binds to cellular protein ubiquitin
• Targets protein for degradation in proteasomes
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Anaplastic Thyroid Cancer
• Associated with mutations of p53
• Inactive p53 protein
• Uncontrolled cell growth
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Cell StructureJason Ryan, MD, MPH
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Endoplasmic Reticulum
• Found in all eukaryotic cells
• Folded membrane of sacs/tubules
• Continuous with nuclear membrane
• Site of synthesis of proteins and lipids
Wikipedia/Public Domain
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RERRough Endoplasmic Reticulum
• Surface of ER covered with ribosomes
• Gives granular or “rough” appearance
• Site of protein synthesis
Wikipedia/Public Domain
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RERRough Endoplasmic Reticulum
• Membrane bound ribosomes• Found in RER
• Produce proteins mostly for secretion from cell
• Protein hormones, digestive enzymes
• Free ribosomes• Found “free” in cytosol
• Produce proteins mostly used by cell
• Metabolism, structure
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Nissl Bodies
• Rough endoplasmic reticulum in neurons
• Synthesize neurotransmitters
Dr. Dimitri Agamanolisneuropathology-web.org
Nissl Bodies
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RERRough Endoplasmic Reticulum
• Abundant in cell that secrete proteins• Goblet cells of intestines (mucus)
• Plasma cells (antibodies)
• Pancreatic beta cells (insulin)
Wikipedia/Public Domain
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SERSmooth Endoplasmic Reticulum
• Portions of ER without ribosomes
• Important for lipid/steroid synthesis
• Also detoxification of drugs and toxins
• Sarcoplasmic reticulum = SER in myocytes• Stores calcium for muscle contraction
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SERSmooth Endoplasmic Reticulum
• Lots of SER found in hepatocytes• Synthesis of cholesterol/lipoproteins
• Many detoxification enzymes
• Cytochrome P450 family of enzymes
• Also found in steroid producing organs• Adrenal glands
• Gonads
Cholesterol
Cortisol Testosterone Estradiol
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Golgi Apparatus
• Proteins leave ER in vesicles→ transported to Golgi
• Fuse with Golgi membrane → empty their contents
• In Golgi → proteins modified
• Sorted for transport to next destination
Wikipedia/Public Domain
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Golgi Apparatus
• Cis Golgi network• Vesicles come into cis face from RER
• Trans Golgi network• Vesicles leave from trans face
• Proteins sorted/shipped by adding signal sequences
OpenStax College
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Golgi Modifications
• Modifies N-oligosaccharides on asparagine
• Adds O-oligosaccharides to serine and threonine
• Adds mannose-6-phosphate to lysosomal proteins
• Likely serves many purposes:• Protects proteins from degradation
• Directs proteins to target location
• Allows protein recognition by receptors
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Oligosaccharides
• Polymers (chains) of sugar molecules
Glucose N-acetyl-glucosamine
Galactose
Mannose
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Oligosaccharides
• N-linked: Attached to nitrogen• Often attached to asparagine (extra N molecule)
• O-linked: Attached to oxygen• Often attached to serine/threonine (extra O molecule)
Asparagine
Serine
Threonine
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N-linked Oligosaccharides
• Synthesized in endoplasmic reticulum
• Sugars added to asparagine (extra N molecule)
• Modified in Golgi apparatus (trimmed, sugars added)
Dna 621/Wikipedia
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O-linked Oligosaccharides
• Occurs in Golgi apparatus
• Sugars added to serine/threonine (extra O molecule)
• Example: Mucins heavily O-glycosylated
OpenStax College
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Mannose-6-Phosphate
• Added to proteins destined for lysosomes• Acid hydrolase enzymes
• Added to N-linked oligosaccharides
• Triggers packaging in trans-Golgi → lysosomes
• Process disrupted/abnormal in I-cell disease
Mannose-6-Phosphate
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I-cell DiseaseInclusion Cell Disease
• Rare autosomal recessive metabolic disorder
• Lysosomal storage disease (mucolipidosis)
• Onset in 1st year of life • Growth failure
• Coarse facial features
• Hypotonia/Motor delay
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I-cell DiseaseInclusion Cell Disease
• Failure of processing in Golgi apparatus• Mannose-6-phosphate NOT found on lysosome proteins
• Deficiency: N-acetylglucosaminyl-1-phosphotransferase
• Phosphate not added to mannose due to missing enzyme
• Result: enzymes secreted outside of cell• Hydrolases missing from lysosomes
• Can be detected in blood/urine (outside cell)
• Lysosomes contain inclusions of undigested glycosaminoglycans and glycolipids
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Endosomes
• Membrane-bound compartments in cells
• Formed by endocytosis• Invagination of plasma membrane to surround molecules
• Pinching off of membrane to form enclosed structure
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Endocytosis
• Receptor-mediated endocytosis• Cells take up specific molecules (ligands) that bind receptors
• Receptors often located in coated pits
• Pinocytosis• Cells ingest droplets of liquid from extracellular space
• Phagocytosis• Cells extends pseudopods
• Encircle particles
• Important part of immune defense
• Macrophages, Neutrophils = professional phagocytes
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Endocytosis
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Endosomes
• Transport contents to lysosome • Often fuse (join together) with membrane of lysosome
• Lysosome digests materials
• Sometimes transport back to cell membrane
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Lysosomes
• Acidic (pH ~4.8)
• Many acid hydrolase enzymes (40+ types)• Require acidic environment
• Breakdown substrates by addition of water molecules
• Breakdown cellular waste
• Also fats, carbohydrates, proteins
• Generate simple compounds
• Returned to cytoplasm to be used by cell
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Lysosomes
• Enzyme deficiency → lysosomal storage disease
• Cellular buildup of macromolecule → disease
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Peroxisomes
• Cellular organelles (membrane-enclosed)
• Contain oxidative enzymes
• Can generate hydrogen peroxide (H2O2)
• Catalase• Oxidizes substances with H2O2
• Detoxifies many substances in liver cells
• Can metabolize ethanol (alternative, minor pathway)
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Peroxisomes
• Beta oxidation fatty acids• Occurs in mitochondria but also peroxisomes
• Peroxisomes preferentially oxidize longer fatty acids
Fatty Acid
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Proteasomes
• Destroy aberrant proteins• Misshaped/misfolded
• Barrel-shaped structure
• Protein “complex”: multiple protein subunits
• Requires ATP
FridoFoe/Wikipedia
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Proteasomes
• Mostly destroys proteins “marked” by ubiquitin• Small protein
• Tags damaged proteins
• May play a role in Parkinson’s disease• Reduced ubiquitin-proteasome activity
• Toxic accumulations of proteins in neurons
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Secretory Pathway
• Series of steps for secretory proteins
• Begins with translation of mRNA in cytosol
• Protein enters endoplasmic reticulum lumen
• Transferred to Golgi
• Exits Golgi in vesicle
• Exocytosis at plasma membrane → secretion
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Signal Sequences
• Found on proteins undergoing synthesis (translation)
• Used to pull free ribosomes to ER membrane• Creates rough ER
• Leads to proteins entry into ER lumen
• Many will ultimately be secreted (via secretory pathway)
• Some will go to ER, other organelles
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Signal Sequences
• Short peptides (proteins)
• Found on N-terminal of protein
• Directs protein-ribosome to endoplasmic reticulum
Amino Acid
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Signal Sequences
• Signal Recognition Particle (SRP)• Ribonucleoproteins found in cytosol
• Complex particle with many proteins and RNA
• Recognize signal sequences
• Moves proteins from cytosol to ER
• SRP Receptor• Found on ER membrane
• Binds SRPs
• Protein translocated through pore into ER lumen
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Coated Vesicles
• Vesicles with protein coat on surface
• Formed from specialized portions of membranes
• Different coats in different forms of traffic
• Important for secretory pathway
• Also important in transport from cell surface
• Three well-characterized coats• Clathrin
• COPI
• COPII
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Clathrin-Coated Vesicles
• Transport between plasma membrane and Golgi
• Also to/from endosomes in cytoplasm
• Major vesicle: receptor-mediated endocytosis• Uptake of extracellular component into vesicle
• Receptors found in “clathrin-coated pits”
• LDL-receptor
• Growth factor receptors
Wikipedia/Public Domain
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COPI and COPII Vesicles
• COPI: Golgi to ER (retrograde)
• COPII: ER to Golgi (anterograde)
OpenStax College
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CytoskeletonJason Ryan, MD, MPH
![Page 288: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/288.jpg)
Cytoskeleton
• System of filaments (Latin = thread)
• All constructed from smaller protein subunits
• Maintains shape of cells
• Moves intracellular traffic
• Pulls chromosomes apart in mitosis
![Page 289: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/289.jpg)
Types of Filaments
• Microfilaments (actin filaments)
• Intermediate filaments
• Microtubules
Microfilaments
Intermediate
Microtubules
7-9nm
10nm
25nm
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MicrofilamentsActin Filaments
• Polymers of protein actin
• Often found under cell membrane
• Many roles: cell shape, cell movement
![Page 291: DNA Replication](https://reader030.vdocuments.pub/reader030/viewer/2022012407/61a82383acb9ad6daf2272c6/html5/thumbnails/291.jpg)
Microvilli
• Extensions of intestinal cell membranes
• Formed from actin filaments
Wikipedia/Public Domain
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Muscle Fibers
• Basic unit: Sarcomere
• Overlapping thin and thick filaments
• Thin filaments: actin and associated proteins
• Thick filaments: myosin
• Myosin filaments slide past actin → contraction
Myosin Actin
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Intermediate filaments
• Maintain cell shape/structure
• Many different types found in variety of cells
• Often used as tumor markers
• Immunohistochemical staining• Antibodies against intermediate filament proteins
• Specific filaments associated with certain tumors
• Various methods for detecting antibody binding
• “Positive staining” suggests tumor origin/type
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Vimentin
• Found in mesenchymal tissue• Cells/tissue derived from mesoderm in embryo
• Mostly connective/soft tissue (i.e. not organs)
• Fibroblasts
• Skeletal muscle
• Mesothelium lining of peritoneum, synovial joints
• Endothelium
• Adipocytes
• Osteoblasts
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Vimentin
• Z-disks in sarcomeres• Contain vimentin and desmin
Myosin Actin
Z disk
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Vimentin
• Sarcoma • Tumor of mesenchymal origin
• Positive for vimentin
• Many subtypes
• Liposarcoma (adipocytes)
• Leiomyosarcoma (smooth muscle)
• Also found in other non-sarcoma tumors• Used to distinguish from other tumors
• Renal cell carcinoma
• Some CNS tumors (meningioma)
• Endometrial carcinoma
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Desmin
• Muscle filament
• Part of Z-disks in sarcomeres (vimentin and desmin)
• Marker for muscle tumors
• Rhabdomyosarcoma
• Leiomyoma and leiomyosarcoma
Myosin Actin
Z disk
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KeratinCytokeratin
• Epithelial cell filaments
• Found in cytoplasm (intracellular)
• Many subtypes (i.e. cytokeratin 8, 18, 19)
• Used to diagnose epithelial tumors (cytokeratin+)
• Useful in squamous cell carcinoma• Cervical cancer
• Head and neck
• Lung
• Skin
• Esophagus
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Lamins
• Forms nuclear envelope• Separates nucleus from cytoplasm
• Outer membrane, inner membrane, intermembrane space
• “Nuclear lamins”
• Note: Laminin = extracellular proteins
BruceBlaus/Wikipedia
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Neurofilaments
• Found in neurons (especially axons)
• Positive staining in many CNS tumors• Neuroblastoma
• Medulloblastoma
• Retinoblastoma
Quasar Jarosz/Wikipedia
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GFAPGlial fibrillary acidic protein
• Major intermediate filament for astrocytes
• Also found in some other CNS glial cells
• Seen in CNS tumors• Astrocytoma
• Glioblastoma
GerryShaw /Wikipedia
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Microtubules
• Polymers of alpha and beta tubulin
• “Heterodimer” units: one alpha, one beta
• Polymerize into a long “protofilament”
• Each dimer has 2 GTP• Alpha GTP: part of structure
• Beta GTP: can be hydrolyzed
Thomas Splettstoesser (www.scistyle.com)Thomas Splettstoesser/Wikipedia
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Microtubules
• Grow from a centrosome near nucleus
• Have a (-) and (+) end
• Emanate in a star pattern in cell
Centrosome
+
-
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Dynamic Instability
• Microtubules grow slowly
• Rapidly disassemble (~100x faster)
• “Dynamic instability”
Thomas Splettstoesser (www.scistyle.com)
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Molecular Motor Proteins
• Bind and move along filaments
• Often carry “cargo”• Organelles (mitochondria)
• Secretory vesicles
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Dynein and Kinesin
• Microtubule motor proteins
• Kinesin moves toward (+) end • Away from nucleus/cell body
• Important for axonal transport (toward terminal)
• Dynein moves toward (-) end• Movement of vesicles
• Localization of Golgi apparatus near cell center
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Cilia and Flagella
• Motility structures
• Built from microtubules and dynein
• Cilia (shorter): Move mucus in respiratory tract
• Flagella (longer): Sperm motility
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Cilia and Flagella
• Microtubules/proteins formed into an “axoneme”
• Structures arranged in special pattern (“9 x 2”)• 9 doublet microtubules in ring
• Surround a pair (“2”) microtubules
Franciscosp2/Wikipedia
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Cilia and Flagella
• Secured by “basal body” root in cell surface
• Nine groups of fused triplets of microtubules
• No central pair
Franciscosp2/Wikipedia
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Cilia and Flagella
• Axonemal dynein: forms bridges between microtubules
• Activated dynein→ pulls on neighboring doublets• Requires ATP (“microtubule dependent ATPase”)
• Sliding of doublets → bending of cilia/flagella
Franciscosp2/Wikipedia
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Cilia
Wikipedia/Public DomainLouisa Howard, Michael Binder
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Primary Ciliary DyskinesiaImmotile-cilia syndrome
• Cilia unable to beat, beat normally, or absent
• Inherited (autosomal recessive)
• Dynein gene mutations
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Primary Ciliary DyskinesiaClinical Features
• Rhinosinusitis• Lining of sinuses irritated, swollen
• Excessive mucus production
• Infertility• Immotile sperm (sperm still viable)
• Dysfunctional fallopian tube cilia (↑ risk ectopic)
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Kartagener’s syndromeManifestation of PCD
• Triad:• Chronic sinusitis
• Bronchiectasis (chronic cough, recurrent infections)
• Situs inversus
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Mitosis
• Chromosomes separate
• Depends on mitotic spindle
• Composed of microtubules
Ali Zifan/Wikipedia
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Microtubule Drugs
• Cancer drugs• Vincristine/Vinblastine (inhibit polymerization)
• Paclitaxel (enhance polymerization – block breakdown)
• Colchicine (gout)• Prevent microtubule assembly
• Disrupts chemotaxis, generation of cytokines, phagocytosis
• Griseofulvin (fungi)
• Mebendazole (helminths)
Pixabay/Public Domain
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Connective TissueJason Ryan, MD, MPH
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Connective Tissue
• Supports/connects organs and other structures
• Key components:• Collagen
• Elastin
• Fibrillin
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Collagen
• Family of fibrous proteins
• Most abundant proteins in human body
• 25% of total protein mass
• Synthesized/secreted by connective tissue cells
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Collagen
• Contains three long α chains
• Basic unit: “triple helix”
• 42 different genes for alpha chains
• Combinations → different collagen types
Vossman
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Collagen
• Large amounts of proline, lysine, and glycine
• Repeating units: Gly-X-Y
Proline Glycine
Lysine
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Collagen Types
• Type I (most common – 90% of collagen)• Bone
• Skin
• Tendons, ligaments
• Cornea
• Internal organs
• Defective production: Osteogenesis imperfecta
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Collagen Types
• Type II • Cartilage
• Intervertebral discs
• Vitreous humor (eye)
• Type III• Skin
• Blood vessels
• Abnormal in some forms of Ehlers-Danlos syndrome
• “Fibrillar collagens”: Types I, II, and III• Collagen molecules assemble into polymers (fibrils)
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Collagen Types
• Type IV• Basement membranes
• Basal lamina (beneath epithelial layer)
• Lens
• Cochlea
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Alport SyndromeHereditary Nephritis
• Genetic type IV collagen defect• Mutations in alpha-3, alpha-4, or alpha-5 chains
• Most commonly X-linked
• Classic triad:• Hematuria
• Hearing loss
• Ocular disturbances
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Collagen Synthesis
• Extensive post-translational modification
• Alpha chains synthesized in rough ER• Contain signal molecules
• “Pre-procollagen”
• Enter ER lumen • Pro-alpha chains
OpenStax College
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Collagen SynthesisEndoplasmic Reticulum Modifications
• Some prolines and lysines are hydroxylated• Form “hydroxyproline” and “hydroxylysine”
• Requires vitamin C (cofactor for hydroxylase enzymes)
• Deficiency of vitamin C → scurvy
• Some hydroxylysines are glycosylated• Sugar molecules added
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Collagen SynthesisEndoplasmic Reticulum Modifications
Lysine
Proline
Hydroxylysine
Hydroxyproline
Hydroxylation(Vitamin C)
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Collagen SynthesisEndoplasmic Reticulum Modifications
Hydroxylysine
Glycosylation
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Scurvy
• Vitamin C deficiency
• Defective pro-alpha chains
• Do not form triple helix
• Degraded in cell (not secreted)
• Fragile blood vessels (bleeding/bruising)
• Loss of teeth
• Loss of wound healing
CDC/Public Domain
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Collagen SynthesisEndoplasmic Reticulum
• Propeptides• Extra amino acids at N and C ends of pro-alpha chains
• Form in fibrillar collagen alpha chains (Type I, II, III)
• Form disulfide bonds that stabilize alpha chains
• Three pro-alpha chains combine: procollagen• Triple helix formation
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Collagen SynthesisExtracellular Modifications
• Moves through Golgi
• Procollagen excreted by exocytosis
• Propeptides (N and C terminal) cleaved
• Tropocollagen formed• Individual triple helix alpha chain molecules
• No propeptides (removed)
• Not yet crosslinked
Vossman
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Collagen SynthesisExtracellular Modifications
• Collagen fibrils form• Tropocollagen much less soluble than procollagen
• Fibrils self assemble
• Strengthened by lysine crosslinking
• Extracellular enzyme: lysyl oxidase
• Requires copper as cofactor
• Collagen fibers: bundles of triple helices
Pixabay/Public Domain
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Crosslinking
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Tropocollagen(no propeptides)
Collagen SynthesisSummary
Pro-alpha chainsHydroxylationGlycosylation
Procollagen(propeptides)
PropeptideRemoval
Collagen Fibrils
EndoplasmicReticulum
ExtracellularSpace
Crosslinking
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Aging Wrinkles
• ↓ production of elastin and collagen in dermis
• Also collagen/elastin fibers thicken and clump
Wikipedia/Public Domain
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SclerodermaSystemic Sclerosis
• Autoimmune disorder
• Stiff, hardened tissue (sclerosis)
• Skin, other organ systems involved
• Caused by fibroblast activation
• Excess collagen deposition
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Osteogenesis Imperfecta“Brittle bone disease”
• Family of genetic bone disorders
• Range of severity (some forms lethal in utero)
• All involve osteoporosis and fractures
• Defective/deficient collagen production
Xiong/Wikipedia
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Osteogenesis Imperfecta“Brittle bone disease”
• Type I: most common form
• Autosomal dominant
• Mutation in COL1A1 or COL1A2 genes• Encode alpha chains for type I collagen
• Abnormal/absent alpha chains
• Triple helix not formed normally
• Decreased production of type I collagen
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Osteogenesis Imperfecta“Brittle bone disease”
• Type II• Lethal in utero
• Type III and IV• More severe than type I
• Severity: II, III, IV, I
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Osteogenesis ImperfectaClinical Features
• Multiple, recurrent fractures with minimal trauma• May be confused with child abuse
• Blue sclera• Clear connective tissue over veins
• Hearing loss• Abnormal malleus, incus, and stapes (ossicles)
Herbert L. Fred, MD and Hendrik A. van Dijk
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Osteogenesis ImperfectaOther Features
• Dentinogenesis imperfecta• Rarely seen in type I
• Common in types III, IV
• Discolored teeth (blue-gray or yellow-brown color)
• Teeth translucent or shiny
• Weak teeth, easily fall out or break
• Bony deformity
• Short stature
BMC Med Genet. 2007 Aug 8;8:52
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Ehlers Danlos Syndrome
• Family of genetic connective tissue disorders• Range of severity
• Range of inheritance patterns
• All caused by defective collagen synthesis
• Predominantly affects joints and skin
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Ehlers Danlos Syndrome
• Classic type• Autosomal dominant (often de novo mutation)
• COL5A1 or COL5A2 genes (type V collagen)
• Type V interacts with other collagens
• Vascular type• Autosomal dominant
• COL3A1 gene (type III collagen)
• Skin, blood vessels
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Ehlers Danlos SyndromeClassic Type (type V collagen)
• Joint hypermobility
• Hyperextensible skin (“velvety” skin)
• Easy bruising
• Thin, wide scars ("cigarette paper" scars)
• Mitral valve prolapse
• Same features in many subtypes (varying degrees)
Piotr Dołżonek/Wikipedia
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Ehlers Danlos SyndromeVascular Type (type III collagen)
• Thin skin, easy bruising
• Rupture of large arteries• CNS (“berry”) aneurysms
• Rupture of “hollow” organs• Intestinal perforation
• Uterus during pregnancy
• Life-threatening form of EDS• 80% have vascular event or rupture by 40 years old
• Median age of death: 48 years old
Wikipedia/Public Domain
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Menkes Disease
• X-linked recessive disorder
• Mutations in the ATP7A gene• ATPase involved in intestinal copper uptake/transport
• Impaired copper absorption → deficiency• Contrast with Wilson’s disease (copper excess)
• Wilson’s ATP7B gene
• ↓ lysyl oxidase activity
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Menkes Disease
• Classic features: Sparse, brittle (“kinky”) hair
• Low body temperature
• CNS features• Hypotonia
• Seizures
• Poor growth
• Developmental delay
• Osteoporosis/fractures
• Usually fatal in childhood
Datta AK, Ghosh T, Nayak K, Ghosh M.
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Elastin
• Connective tissue protein
• Main component of elastic fibers• Allows stretching/recoil
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Elastin
• Arteries• Dominant elastic protein
• Makes up 50% of aortic tissue
• Skin
• Lungs
• Ligaments
• Vocal cords
• Spinal ligaments (ligamenta flava)
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Elastin
• Contains glycine, lysine, and proline (like collagen)
• Mostly non-hydroxylated amino acids• No hydroxylysine
• Some hydroxyproline (less than collagen)
• Not glycosylated
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Elastin
• Secreted as tropoelastin
• Assembled into elastin fibers with crosslinking
sportEX journals/Flikr
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α1 Anti-trypsin Deficiency
• Inherited (autosomal co-dominant)
• Decreased or dysfunctional AAT
• Inhibitor of enzyme elastase
• Excessive breakdown of elastin
• Result: Emphysema
• Lung damage
• Imbalance between neutrophil elastase (destroys elastin) and elastase inhibitor AAT (protects elastin)
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Williams SyndromeWilliams-Beuren syndrome
• Partial deletion on long arm of chromosome 7
• Deleted portion includes gene for elastin
• Elfin appearance, intellectual disability
• Supravalvular aortic stenosis • Constriction of ascending aorta above aortic valve
• High prevalance among children with WS
• Histology: Loss of elastin
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Fibrillin
• Glycoprotein
• Major component of microfibrils
• Sheath that surrounds elastin core
• Elastic fibers: Elastin, microfibrils, other molecules
• Abundant in the aorta
• Deficient fibrillin: Marfan syndrome
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Marfan Syndrome
• Genetic connective tissue disorder
• Abnormal fibrillin
• Mutations in FBN1 gene (chromosome 15)• Codes for fibrillin-1
• Affects bones, joints, heart, eyes
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Marfan Syndrome
• Classic appearance: Tall with long wingspan
Wikipedia/Public Domain
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Marfan Syndrome
• Classic finding: Pectus Excavatum (sunken chest)
Wikipedia/Public Domain
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Marfan Syndrome
• Extremities: • Hypermobile joints
• Long fingers and toes
• “Arachnodactyly”: Long, curved finger (like a spider)
• Wrist sign: • Tip of thumb covers entire fingernail of fifth finger
• Thumb sign:• Thumb protrudes beyond ulnar border
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Marfan Syndrome
Wikipedia/Public DomainPixabay/Public Domain
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Marfan SyndromeEye
• Cataracts at early age (“pre-senile”)
• Dislocation of lens• Commonly due to trauma
• Can be associated with systemic condition
• Marfan most common
• Classically upward/outward lens dislocation
Retina Gallery
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Marfan SyndromeCardiovascular
• Mitral valve prolapse
• Thoracic aortic aneurysms and dissection• Cystic medial necrosis
• Cysts and necrosis in medial layer
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Marfanoid Habitus
• Tall with long wingspan
• Long fingers
• Seen in some rare systemic disease• Homocystinuria
• MEN 2B
• Rare forms of Ehlers Danlos