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DNA replication
醫技系 楊孔嘉
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References
1. Nature 2003, 421:p431 (replication & recombination)2. EMBO reports 2003, 4:p666 (replication) 3. BioEssays 2003, 25:p116 (MCM proteins)4. Gene 2003, 310:p1 (ORC cycle)5. FEMS Microbiol Rev 2003, 26:p533 (structure of OBPs)6. Cancer Lett 2003, 194:p139 (telomerase)7. J Struct Biol 2003, 140:p17 (initiation)8. EMBO reports 2003, 4:p1 (kinase)9. Curr Opin Microbiol 2003, 6:p146 (cell size)10. Genome Biol 2003, 4:p204 (microarray)11. Annu Rev Biochem 2002, 71:p333 (eukaryotes)12. Nature Cell Biology 2004, 6:p648 (origin firing)13. Acta Biochimica Polonica 2005, 52:p1 (SSB)14. GENES VIII chapter 14 (Introduction)
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Learning objectives
Let the students know where we are and where to go about DNA replication.
Prokaryotic cells Eukaryotic cells
the same or different ?
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“ This structure has novel features which are of considerable biological interest ”
Nature 171, April 25 (1953) p. 737-738 - by James Watson and Francis Crick
Molecular structure of nucleic acid
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The principles of DNA replication
template dependent requires the four dNTPs primer-dependentsynthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'
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The principles of DNA replication
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OrganismGenome
size (bases)
Estimated genes
Human (Homo sapiens)* 3200 x 106 20,000-25,000
Laboratory mouse (M. musculus)
2600 x 106 20,000
Mustard weed (A. thaliana)
100 x 106 25,000
Roundworm (C. elegans) 97 x 106 19,000Fruit fly (D. melanogaster) 137 x 106 13,000Yeast (S. cerevisiae) 12.1 x 106 6,000Bacterium (E. coli) 4.6 x 106 3,200Human immunodeficiency virus (HIV)
~9700 9
Hepatitis C virus (HCV) ~9030 1SARS-associated Coronavirus
~29,700 ~11
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The central questions to be answered
(1) Does DNA synthesis begin at a defined place ?
(2) What determines replication initiation sites ?
(3) What regulates an origin to fire once and only once per cell cycle ?
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FeatureProkaryotic (E. coli)
Eukaryotic (human HeLa cells in culture)
DNA content (bp)
4.6 x 106 3.2 x 109
DNA replication rate (nt/sec/rep/ fork)
850 60-90
Number of replication origins/cell
1 103-104
Hours for complete genome replication
0.67 8
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Chronicle for DNA replication
1950 1960 1970 1980 1990 2000
In 1972, Kornberg et al. demonstrated in vitro enzymatic activity of DNA polymerase from E. Coli
J. Biol. Chem. 1972;247:p241.
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Chronicle for DNA replication
1950 1960 1970 1980 1990 2000
In 1984, the first mammalian-based DNA replication system (SV40) that initiated DNA synthesis successfully in vitro was developed.Proc Natl Acad Sci U S A. 1984;81:p6973.
In 1985, Greider and Blackburn identified enzymatic activity of the telomerase in Tetrahymena extract Cell 1985;43:p405.
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Chronicle for DNA replication
1950 1960 1970 1980 1990 2000
Establish the molecular mechanism of DNA replication, involving the stages of initiation, priming, elongation and termination
Establish the linkage of DNA replication to cell cycle control
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Study on DNA replication
1. E. Coli model2. Yeast genetic model3. Simian virus 40 (SV40) model4. Adenovirus model5. Herpes simplex virus (HSV) model
Genetic approach: temperature sensitive(ts) mutants
Biochemical approach: in vitro complementation assay
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template dependent requires the four dNTPs primer-dependentsynthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'
The principles of DNA replication
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Priming for DNA replication DNA polymerases is incapable of initiating
DNA polymerization in the absence of an existing 3' -OH
The priming RNA supplies the necessary 3' -OH group to prime DNA polymerization and is later clipped off and replaced with DNA by the DNA polymerase
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Priming for DNA replication
Due to low fidelity of RNA polymerases, primase and primosome takes over for priming RNA synthesis.
Okazaki fragment formation requires repeated initiation of DNA replication de novo.
Three kind of priming: an RNA primer, a nick in DNA, a priming protein
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Priming for DNA replication
RNA primer:The majority of prokaryotic and eukaryotic DNA replication
DNA primer: nick translation, hairpin loop
Protein (terminal protein + dCTP) as a primer:adenovirus DNA replication
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template dependent requires the four dNTPs primer-dependentsynthesizes DNA in the 5' to 3' direction, reading the template 3' to 5'
The principles of DNA replication
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energy source
energy source
Why DNA synthesis is 5’ to 3’ ?
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Replication complex and replication forkBi-directional DNA replicationEnzymology of DNA replication
The principles of DNA replication
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The DNA replication fork
Lagging strand with Okazaki fragments (1000-2000 base)
Leading strand
Most recently synthesized DNA
DNA replication fork
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Replication complex and replication forkBi-directional DNA replicationEnzymology of DNA replication
The principles of DNA replication
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Directions of DNA replication
Directions of DNA replication
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Experimental evidence of bidirecitonal DNA replication
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Large eukaryotic DNA has multiple origins of replication
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Replication complex and replication forkWhy DNA synthesis is 5’ to 3’Bi-directional DNA replicationEnzymology of DNA replicationpolymerase, primase, helicase, single strand DNA binding protein (SSB), ligase, topoisomerase, telomerase
The principles of DNA replication
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Prokaryotic system
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Characteristic
Polymerase I
Polymerase II
Polymerase III
gene polA polB polC
MW 103,000 90,000 130,000
#molecules/cell 400 100 103‘ exonuclease
yes yes yes*
5‘ exonuclease
yes no no
Biological function
DNA repair, RNA primer
excision
SOS DNA repair
Replicative chain growth
DNA polymerase of E. coli
*Polymerases IV and V participate in SOS repair function.
Proofreading function
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E. Coli DNA Pol III (replicative polymerase)
complex enzyme with ten subunits
subunit contains the active site for nucleotide addition
subunit is a 3’ to 5’ exonuclease, a proofreading activity that removes incorrectly added bases
subunit can stimulate exonuclease
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E. Coli DNA Pol III (replicative polymerase)
subunit is a processivity factor -> prevents enzyme from falling off prematurely thus decrease likelihood of frameshift mutation
subunit is a clamp loader -> places the processivity subunit on DNA
subunit is a dimerizing subunit -> link the two catalytic cores together
Note: In mammalian system: PCNA is a processivity factor, RF-C is a clamp loader
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Finger Thumb
Palm
DNA
3‘-5’ exonuclease
PDB: 1D8Y
3D structure of DNA polymerase
nucleotideRotate 60
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Editing mode Polymerizing mode
3D structure of DNA polymerase
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-subunit clamp
subunit
subunit
DNA
waterwater
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Assembly of E. Coli DNA Pol III
Clamp loader () cleaves ATP to
load clamp () on DNA
Enter the core enzyme ()
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Assembly of E. Coli DNA Pol III
Dimerization by subunits
Repetitively loading on the lagging strand
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Stages of DNA replication
Initiation Elongation Termination
Note: At the initiation stage, origin binding proteins (OBP) and ATP is required to trigger the following events • Melt the two strands of DNA• Recruit protein factors essential for replication
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Origin for DNA replication in E. coli
245 bp ori C region
Seven replication factors working at the origin-DnaA: origin binding protein (OBP)-DnaB: helicase-DnaC: recognition protein-HU: bend DNA structure-Gyrase: rotate one strand over the other -SSB: single strand binding -DnaG: primase
13 bp sequences DnaA-binding sites (3 repeats) (4 repeats of 9 bp)
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3D structure of DnaAATPases Associated with various cellularActivities (AAA+)
DBD
Mg2+ ADP cofactor
Note: Common features are shared in the initiator of chromosomal replication in bacteria, archaea and eukaryotes FEMS Microbiology Review 2003, 26, p533
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DnaA
Initiation of DNA replication in E. coli
DnaB
DnaB
DnaBDnaB
DnaG
DnaG
A-T rich region
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Origin for DNA replication in E. coli
Prepriming complex Replication bubble
DnaA
DnaB
DnaB
DnaBDnaB
DnaG
DnaG
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Dna A
Regulatory inhibition of DnaA
DnaA protein undergoes conformation change in hydrolysis of ATP and unable to initiate further replication rounds
Note: DnaA protein can be reactivated either by acidic phospholipids or DnaK chaperon, that exchange ADP by ATP
DnaB
DnaB
DnaBDnaB
DnaG
DnaG
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Competition model for DnaA-ATP and DnaA-
ADP: coupling the initiation of chromosome replication to cell size Current Opinion in Microbiology 2003, 6:p146
eclipse
DNA replication
Cell growth
Total DnaA-ADP (inactive)
Total DnaA-ATP (active)
Old DnaA-ATP (active)
New DnaA-ATP (active)
completion of cell division
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The factors that regulate an origin to fire only once per cell cycle
Convert DnaA-ATP to DnaA-ADP Repress DnaA transcription
Prevent DnaA binding -> DNA replication
-> Hemimethylated GATC at the oriC DNA-> sequestered to plasma membrane
-> thus a membrane-bound inhibitor prevents re-initiation of the origin by DnaA
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DNA helicase
~ 60 bp
DNA helicase
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Single strand binding protein (SSB)
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Single strand binding protein (SSB)
The SSBs from prokaryotes and eukaryotes share a common core ssDNA-binding domain
Application of SSB in biotechnology
• Increase amplification efficiency in single PCR
• Prevention of primer dimer formation in multiplex PCR
• Increased size of cDNA in RT-PCR
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Replication complex and replication fork
Leading strand template
Lagging strand template
Leading strand
Okazaki fragment
Dimer of Pol IIIPrimase
Single strand binding protein (SSB)
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Lagging strand synthesis in E. Coli
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Characteristic
Polymerase I
Polymerase II
Polymerase III
gene polA polB polC
MW 103,000 90,000 130,000
#molecules/cell 400 100 103‘ exonuclease
yes yes yes*
5‘ exonuclease
yes no no
Biological function
DNA repair, RNA primer
excision
SOS DNA repair
Replicative chain growth
DNA polymerase of E. coli
Note: In mammalian system: RNAse HI and FEN1 remove the RNA primer
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The replication termini in E. Coli
Binding of tus protein -> stop DnaB helicase activity
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Wake up
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Eukaryotic system
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Eukaryotic model system
1. Yeast2. Simian virus 40 (SV40)3. Early embryos of Drosophila
and Xenopus 4. Mammalian cells5. Adenovirus
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Polymerase Location
Size of catalytic subunit
(kD)
Biological function
Alpha ()/primase
nucleus 160-185Priming &
Lagging strand replication
Delta () nucleus 125Leading strand
replication
Epsilon nucleus210-230 or
125-140
Lagging strand replication & DNA repair
Beta () nucleus 40 DNA repair
Gamma ()mitochondri
a125 Mitochondrial
DNA replication
DNA polymerase of eukaryotic cells High fidelity enzyme
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Polymerase LocationBiological function
Zeta () nucleusThymine dimer
bypass
Eta () nucleus Base damage repair
Iota () nucleus Required in meiosis
Kappa () nucleusDeletion & base
substitution
DNA polymerase of eukaryotic cells
Low fidelity enzyme
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06_08_monitorprogress.jpg
DNA microarrays can be used to locate DNA replication origins
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Species Replication origin
S. cerevisiaeCore A + B1, B2, B3 elements (~50
bp) (autonomously replicating sequence, ARS)
S. pombe spread over 800-1000 bp
Early embryos of Drosophila and Xenopus
Little or no sequence specificity
Drosophila follicle cells
Specific cis-acting elements, not yet defined sequences
Human -globin
Specific cis-acting elements, not yet defined sequencesNote: AT-rich sequences might be required at the
replication origin
Replication origin of eukaryotic cells
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Mitochondrial DNA replication starts at a D-loop
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Where does DNA replication take place ?
S phase
0 h 5 h 8 h 12 h 15 h
Tracing by (1) biotin-labelled dUTP (2) bromodeoxyuridine (BrdU) (3) GFP-tagged proteins (GFP-PCNA)
C2C12 mouse myoblast cell
J Cell Biol. 2000 Apr 17;149(2):271-80.
Proliferating cell nuclear antigen
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Where does DNA replication take place ?
S phase
0 h 5 h 8 h 12 h 15 h
early S-phase mid S-phase late S-phase
Note: DNA replication takes place at replication foci in the Nucleus, where the replication factors are assembled.
J Cell Biol. 2000 Apr 17;149(2):271-80.
nucleoli
Replisome within a replication focus
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Features of DNA replication in eukaryotes
Initiation: licensing and activationformation of pre-replication complex (pre-RC) -> initiation of replication complex (RC)
Elongation Termination: telomere and telomerase
Note: The origin binding proteins (OBP), forming origin recognition complex (ORC), are required for assembly of the pre-RC.
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Licensing and activation of DNA replication
origin
Licensing factors binding
Note: The DNA replication events of licensing and activation are coordinated with cell cycle progress. BioEssays 2003, 25: p116
M/G1
Early S
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molecular combing and fiber spreading studies
Digoxigenin-dUTP is added at t = 0, and biotin-dUTP is added at t = 10, 20, or 30 min after the release
Note: Linear macromolecule, like DNA, which can be anchored on lysine-coating surface by their
extremities only.
10’
20’
30’
early fork
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Distribution of eye-to-eye distances (ETED)
Note: the ETED was measured by electron microscopy, molecular combing and fiber spreading studies
Sperm nuclei replicated in Xenopus egg extracts
10 kb
J Mol Biol 2002;320:p741
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Licensing and activation of replication origins
BioEssays 2003, 25: p116
Licensing factors:ORC (origin recognition complex) 1-6, CDC6, CDT1
G1/S transition: CDC7 and sCDK activation ----->(1) Loading the DNA polymerase and DNA replication progresses(2) Releasing of MCM2-7
Note: reloading of MCM2-7 is prevented by CDKs and geminin until cells pass through mitosis
G1 phase
S phase
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Licensing and activation of replication origins
BioEssays 2003, 25: p116
Note: the ORC binding site and replication origin (AT-rich repeats) locate as neighbors but not the same site
Inactivation of other MCM complexesAT-rich repeats
ORC 1-6, CDC6, CDT1M/G1 phase
S phase
G1 phase MCM2-7
CDC45
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ATR and ATM regulate the timing of DNA
replication origin firing Nature cell biology 2004, 6, p648
ATM: Ataxia telangiectasia mutated ATR: Rad3-related-kinase
ATM/ATR feedback
+ caffeine to inhibit ATR/ATM
DNA damage -> activate ATM/ATR
-> down-regulate cdc7/cdk2 -> Inhibit origin firing
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ORC cycle in eukaryotic DNA replicationGene 2003, 310:p1
Note: the ORC cycle restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cycle binding site
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3D structure of origin binding protein (OBP)
AAA+ superfamily
DnaADnaA CDC6 CDC6
FEMS Microbiology Review 2003, 26, p533
Mg2+ ADP cofactor Mg2+ ADP cofactor
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Phylogenetic tree analysis of ORBs Based on amino acid sequences
FEMS Microbiology Review 2003, 26, p533
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Simian virus 40 DNA replication in vitro
Li JJ, Kelly TJ.
A biochemical assay for the replication activity -Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with SV40 -Catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. -Extracts prepared from uninfected monkey cells also support replication only in the presence of added SV40 large tumor (T) antigen
-The in vitro replication reaction proceeds via branched intermediates (theta structures).
Proc Natl Acad Sci U S A.
1984;81:p6973.
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Re-constitution of purified components of the DNA synthesome
Study of simian virus 40 DNA replication in vitro
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replication origin
SV40 T-Ag-Initiator: binds to the origin
-Helicase: unwinds duplex DNA
-RPA: single-stranded DNA binding protein
-DNA polymerase /primase
- Replication factor C (RF-C): clamp loader
protein
-Proliferating cell nuclear antigen (PCNA):
a general recruiting factor/processivity factor
a. displace pol- by Pol- or Pol-
b. recruit Fen 1 (exonuclease) and Lig 1 (ligase)
-Pol : DNA elongation
RNA-DNA primer synthesis
SV40 DNA replication in vitro
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-SV40 T-Ag: binds to the origin/unwinds duplex DNA
-RPA: single-stranded DNA binding protein
-DNA polymerase /primase: RNA primer synthesis
-PCNA/RFC: release DNA polymerase
-Pol : DNA elongation
DNA-RNA primer
SV40 T-Ag/helicase
SV40 DNA replication in vitro
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-RNase H: remove RNA primer
-FEN1 (5’-3’ exonuclease): remove RNA primer
-DNA ligase I: joins the end of the Okazaki fragments
DNA-RNA primer
SV40 T-Ag/helicase
SV40 DNA replication in vitro
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Eukaryotic DNA replication synthesome
At least two different DNA polymerases ( and )A single-stranded DNA-binding protein (RPA)A clamp-loading complex (RFC, PCNA)A polymerase clamp combine to replicate DNA
Annu Rev Biochem 1998;67:p721
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Core proteins at the DNA replication forkNature, 2003, 421, p431
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Eukaryotic DNA replication synthesome
ori
distributive mode: pol processive mode: pol and (RF-C-independent) (RF-C-dependent)
oriPCNA
PCNARF-C
Primosome complex
RF-C
EMBO J 2002;21:p2485
inhibitor to pol
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Isolation of replication competent complex from nucleus
Chromatography
Nuclear extract
Protein identification by westernin the high-molecular weight RC complex
EMBO J 2002;21:p2485
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Gel filtration
Nuclear extract
Protein identification by western in the high-molecular weight RC complex
Note: RC complex consists of (1) replication factory (pol , pol , RF-C, ligase I, topo I, RP-A)(2) cell cycle regulators
Isolation of replication competent complex from nucleus
EMBO J 2002;21:p2485
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Cell cycle progress and checkpoint control
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Pol activity Pol activity RC compositionRC composition
EMBO J 2002;21:p2485
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CDK-driven replication switch model
Replication factory (replication synthesome)
- pol , pol , RF-C, ligase I, topo I, RP-A
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Dynamic of the DNA synthesome
At G1/S transition, DNA synthesome are
maintained as chromatin-free
At S phase, there are two forms of DNA
synthesome
At G2 phase, DNA synthesome are
maintained as chromatin-free EMBO 2003, 4, p666
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Phosphorylation of the DNA synthesome
Phosphorylation of Pol-/primerase (early)1. Pre-replication complex
-> replication complex2. Inhibition of the re-initiation of
replication origin
Phosphorylation of ligase I(late)1. Response to DNA damage
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DNA replication factory (DNA synthesome)
EMBO 2003, 4, p666
Replication foci
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Assorted replication factors
DNA replication DNA repair DNA recombination Chromatin remodelling
- Replication protein A - DNA polymerase-- DNA polymerase- - DNA ligase I- DNA topoisomerase II- - DNA-C5-methyltrasferase- Histone deacetylase - Chromatin assembly factor- MSH3/MSH9 - hMTH- MRE11 - Cdk2/Cyclin A
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Chromatin dynamics at DNA replication fork
Eur. J. Biochem 2004, 271, p2335
HAT: histone acetyltransferaseCR: chromatin remodeling machinery
helicase
DNA replication
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Telomeres
AGGGTT AGGGTT AGGGTT 3‘
TCCCAA 5‘Newly synthesized tagging strand
Telomere repeats
Telomeres consist of several hundred to a few thousand base pairs composed of repetitive oligomeric sequences with a high G contents (e.g. AGGGTT)
3’ end of G-rich strand extends in single-stranded form 10-16 nucleotides beyond 5’ end
Telomeric sequences differ depending on the organism but are in each case generated by the telomerase enzyme
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Telomerase enzyme
telomerase RNA
Note: The telomerase enzyme consists two parts: telomerase reverse transcriptase (TERT)and telomerase RNA (TR)
Cancer letter 2003, 194, p139
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Telomerase mechanism of action
Telomerase reverse transcriptase
Telomerase RNA
Note: the G-strand extension, telomere elongation and DNA replication may occur contemporaneously
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telomerase RNA
Mutations of telomerase RNA is associated with an autosomal dominant variant of dyskeratosis congenita
Note: Patients exhibit reduced levels of the telomerase RNA and shortened telomere when the 3’ end TR is mutated
Nature 2001;413:p432
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(1) The function of telomerase is to compensate for the shortening of telomeres that occurs at each replication cycle. (2) Telomerase is turned off in many somatic cells, typically when differentiation occurs. Continued division in cells that lack telomerase activity will cause the telomeres to shorten in each generation. The cells are not capable to continue propagation once telomere lengths have become too short. (3) When cells enter senescence as the result of telomere shortening, p53 is activated, leading to growth arrest or apoptosis. (4) The telomerase activity usually is reactivated in tumors. The process of immortalization might be involved the reactivation of telomerase.
Telomerase support for continued cell division
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Proteins associated with human telomerase activity
TEP1 non-steiochiometric association with activity
Stau binds hTR L22 binds hTR Dyskerin binds hTR P23/p90 binds amino terminus of hTERT hnRNPs associated with activity; affect
telomere length
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The central questions to be answered
(1) Does DNA synthesis begin at a defined place ?
(2) What determines replication intiation sites ?
(3) What regulates an origin to fire once and only once per cell cycle ?
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The endThe end
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Questions
1. How to regulate an origin to fire once and only once per DNA replication cycle (prokaryotes & eukaryotes) ?
2. How the DNA telomerase work ? Describe the biological significance of DNA telomerase in DNA replication.
3. How to coordinate DNA replication and cell cycle in eukaryotes ?