bc34lec3 nucleic acids

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Inhibitors of the Genetic

Mechanisms

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Macrolides

•  Erythromycin is a naturally-occurring

macrolide derived from Streptomyces

erythreus

•  Structural derivatives include clarithromycin and azithromycin:

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Macrolide Structure

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Macrolide binding in thecrytallographic structure of H.

marismortui 50S ribosomal subunit

http://www.ncbi.nlm.nih.gov/pubmed/11698379 

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 Aminoglycosides

•  Initial discovery in the late 1940s, withstreptomycin being the first used; gentamicin,tobramycin and amikacin are most commonly usedaminoglycosides

•   All derived from an actinomycete or aresemisynthetic derivatives

•  Consist of 2 or more amino sugars linked to an

aminocyclitol ring by glycosidic bonds =aminoglycoside

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 Aminoglycoside Structure

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 AminoglycosidesMechanism of Action

•  Multifactorial, but ultimately involvesinhibition of protein synthesis

• Irreversibly bind to 30S ribosomesdisrupting the initiation of proteinsynthesis, decreases overall proteinsynthesis, and produces misreading of 

mRNA•  Most are bacteriocidal

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Streptogramins

•  Example: Synercid® 

•  synthetic pristinamycin derivatives in a

30:70 w/w ratio - Quinupristin:Dalfopristin

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Linezolid

Mechanism of Action

•  Binds to the 50S ribosomal subunit near tosurface interface of 30S subunit – causes

inhibition of 70S initiation complex whichinhibits protein synthesis

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Clindamycin

Clindamycin is a semisynthetic derivative of 

lincomycin which was isolated from

Streptomyces lincolnesis in 1962 

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Clindamycin

Mechanism of Action

Ø Inhibits protein synthesis by binding

exclusively to the 50S ribosomal subunit

§  Binds in close proximity to macrolides –

competitive inhibition

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Tetracycline

•  Tetracyclines inhibit

bacterial protein

synthesis by blocking

the attachment of thetransfer RNA-amino

acid to the ribosome.

More precisely they

are inhibitors of thecodon-anticodon

interaction.

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Metronidazole

Metronidazole is a synthetic nitroimidazole

antibiotic derived from azomycin. First found

to be active against protozoa, and then

against anaerobes where it is still extremelyuseful. 

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Metronidazole

Mechanism of Action

Ø Ultimately inhibits DNA synthesis 

§  Selective toxicity against anaerobic and

microaerophilic bacteria due to the presence of ferredoxins within these bacteria

§  Ferredoxins donate electrons to form highlyreactive nitro anion which damage bacterial

DNA and cause cell death

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Summary of antibiotic action on the

genetic mechanism

http://hstalks.com/main/view_talk.php?t=798&r=285&c=252

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Mutation and genetic

disorders

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Mutation

• 

Hereditable change in DNA resulting from change innucleotide sequence

•  Types of mutation –  Point mutations

•  Swap one base for another; insert a base; delete a base

 – Macrolesions•  Alteration of the number of copies of a short repeat and

•  Large insertions into a gene

•  Multiple causes –  Spontaneous

• DNA replication/repair errors•  Insertion of transposons

 –  Mutagens (physical, chemical, viral)

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DNA Mutations: An Overview

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Point Mutations•  Single or few base pair changes

•  Provide “background rate” of mutation –  critically important to evolution

•  More likely to lead to loss of function than gain of function

• 

Causes point mutation –  Induced•  action of mutagen; environmental agent that alters nucleotide

sequence

•  process of inducing mutations by mutagens called mutagenesis

 – Spontaneous•  arise in absence of known mutagen

•  may be due to tautomerism

•  may be caused by errors in DNA replication

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Types of point mutation

•  Base substitution

 –  transition•   A↔ G (purine↔ purine) (A·T↔ G·C)

•  C↔ T (pyrimidine↔ pyrimidine) (C·G↔ T· A)

 –  transversion

•  purine↔ pyrimidine (e.g., A↔ C) (A·T↔ C·G)

•   Addition or deletion of nucleotide pairs (base-pair addition or deletion) –  also called indel mutations

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Types of mutation and effects

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Functional consequences

•  Missense mutations differ in severity –  conservative AA substitution substitutes chemically

similar AA; less likely to alter function

 –  nonconservative AA substitution replaces chemically

different AA; more likely to alter function•  Nonsense mutation results in premature termination

of translation

 –  truncated polypeptides often are nonfunctional

•  Point mutation in non-coding region may or may not

have visible effect (e.g., regulatory region)

S t M t ti

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Spontaneous Mutation:

Tautomeric shift

•  Natural variation in chemical form of base(isomers); differ in position of atoms & bonds –  amino (normal)↔ imino (rare)

 –  keto (normal)↔ enol (rare)

•  Results in mutation during DNA replication –  base in rare tautomeric form pairs with chemically

similar base of its normal complement

 –  e.g., C·G↔ C*·G at replication results in C*· A whichresolves to C· A upon reverse of tautomeric shift

 –  at next DNA replication event, use of A strand resultsin T· A base pair, a transition

•  Contributes to spontaneous mutation rate

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Common Tautomers & Base-

pairingThe more common ‘keto’ and ‘amino’ form of eachbase

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Rare tautomers

base-pair differentlythan the morecommon tautomers

T(enol) pairs with G

G(enol) pairs with T 

C(imino) pairs with A

A(imino) pairs with C

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Tautomeric shifts can be a source of rare spontaneous mutation

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During the next round of DNA replication, the transitionmutation becomes “fixed” in the DNA.

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Creation of indel mutations

The slippage model for creation of insertions and deletions

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Induced mutation: Physical Agents

•  Ultraviolet radiation – Thymine is most susceptible; UV radiation

generates thymine diradicals which can

dimerizes (pyrimidine-pyrimidine dimers) – activates SOS system, resulting in insertion of 

incorrect base

•  Ionizing radiation

 – Generates free radicals from cleavage of molecules including H2O, bases, sugar-phosphate backbones

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Formation of the most toxic and mutagenic DNA lesion, cyclobutane-pyrimidine dimers by UV radiation.

Dimers can form between two adjacent pyrimidines. (A) thymine-thyminecyclobutane-pyrimidine dimer, and (B) thymine-cytosine dimer and their 

photoreactivation by the enzyme photolyase in the presence of light.

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UV light inducesthe formation ofcyclobutane

dimers(pyrimidinedimers) and 6-4photoproducts

These products

cause significant‘kinks’ in theDNA and stopreplication andtranscription

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Free-radical effects

•  Oxidative damage to bases

 – caused by superoxide and peroxide radicals

 – chemically alter base pairing properties

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Oxidatively Damaged Bases

Active oxygen species like superoxide radicals (O2), hydrogenperoxide (H2O2) and hydroxyl radicals (OH.) are produced byaerobic respiration and can damage DNA

Pairs with A

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Induced Mutation: Chemical agents

•  Alkylating agents

•  Intercalating agents

• Deaminating agents

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 Alkylating Agents

•  Depurination, spontaneous loss of G or A

•  agent modifies base resulting in altered

pairing•  e.g., EMS (ethyl methanesulfonate) and NG

(nitrosoguanidine), formaldehyde, CHCl3, epoxides

•  results in replication block and insertion of nonspecific bases by SOS system

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Depurination occursmore often than youmight think

Mammalian cell losesabout 10,000 purinesfrom its DNA in a 20hour cell generationperiod

Obviously, theselesions must berepaired

Spontaneous Depurination

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 Alkylating agent: substitutions

•  Mutagens have different mutational

specificity

 – Base analogs

•  similar to nitrogenous bases of DNA, but havealtered pairing properties

•  e.g., 5-bromouracil (5-BU) and 2-aminopurine (2-AP)

•  result in transitions

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 Action of 5-BU5-BU can frequently change to either the enol or ionized form

Causes G-C to A-T or A-T to G-C transitions

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 Action of 2-APSimilar to 5-BU but with a different specificity

A-T to G-C transitions and G-C to A-T transitions

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Intercalating agents

•  flat, planar molecules intercalate between

base pairs, disrupt DNA synthesis

 – e.g., proflavin, acridine orange, aflatoxin,

benzo(a)pyrene

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Intercalating agentscan cause single baseadditions and deletions

R ti I t l ti

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Reactive Intercalation:

 Aflatoxin

Aflatoxin modifies guanine andresults in loss of base in DNA

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Deamination

•  Deamination, converts cytosine to uracil

which pairs with adenine at replication

•  Indel mutations

 – result in translation frameshift

 – often occur in regions of repeated bases

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Deamination of C yields U whichbase pairs with A

Results in C-G toT-A transition

This occurs

spontaneously

Deamination

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Hotspots for mutation often reflect an aspect of the DNAsequence, for example, a sequence repeat

FS5, FS25, FS45, FS65 result from addition of one set of CTGG which isrepeated three times in the lacI gene

FS2, FS84 result from loss of one set of CTGG

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The Ames test was developedby Bruce Ames for evaluatingvarious chemicals for theirpotential as mutagens andcarcinogens

The Ames Test

1. Chemical injected into

mouse where it is metabolized

2. Liver (site of most chemicalmetabolism) is isolated and anextract is made that containsthe metabolized chemical

3. Extract is tested for mutagenicityin 3 strains of Salmonella to score forvarious kinds of mutations

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Defense mechanisms

•  Free radical scavengers

 – Vitamins A, E, C; only C is water soluble and

does not accumulate in the body

 – GSH (glutathione) converted to GSSG

 – Metallothionein (61 amino acids with 20

cysteines and 7 zinc atoms

•  Enzymes – Photolyase: photoreactivation repair 

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Photoreactivation repair 

•  Various enzymes with specific properties,

e.g. cleavage of pyrimidine dimers (T^T)

 – enzyme binds to dimer 

 – energy of blue light cleaves bond

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Defense mechanisms: enzymes

 – Demethylase (suicide enzyme)

 – Superoxide dismutase (converts superoxides

to H2O2)

 – Catalase (converts H2O2 to H2O + O2

 – Peroxidases

•  Repair systems

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Defense mechanisms: DNA Repair 

•  DNA damage must be repaired

•  Damaged DNA in any cell will be deleterious to

cellular function

 – in germ line cells mutations heritableBacteria are particularly prone to damage

 – haploid all mutations are dominant

 – unicellular whole organism affected

•  Repair most effective before replication at site

•  Repair can be more or less error-prone

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Types of Repair: Mismatch

•  Mismatch repair genes: mut 

•  Mismatched bp detected

•  Nearby ss cut and excision of ssDNA

past mismatch

•  – daughter strand preferentially excised

•  DNA polymerase repairs gap

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 Apurinic gap repair 

•   Apurinic gaps

 – most common spontaneous degradation of 

DNA

 – hydrolysis of sugar-purine bond - loss of purine(A, G)

•  Repair by AP endonuclease

 – removes base, ss gap filled by polymerase•  If no repair then A inserted at replication

 – G-C → T-A transversion mutation

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Excision repair 

•  Multi-enzyme system

•  Repairs stretch of damaged nt’s

 – damaged ssDNA excised

 – gap repair by polymerase

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Nucleotide-excision

repair removes bulky

DNA lesions that

distort the doublehelix

http://www.nature.com/

scitable/resource?action=showFullImageF

orTopic&imgSrc=36086/pierce-17_30_FULL.jpg 

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Post-replication repair 

•  Polymerase can’t replicate acrossdamaged nt’s

•  Leaves a ss gap

•  Gap filled by strand exchange from theother dsDNA

•  Secondary gap repaired by polymerase