Chapter 9 The Mutability and Repair of DNA

1.replication errors and their repair2. DNA damage3.Repair of DNA damage

Outline:

Section 1:replication errors and their repair

Important definations: Transitions:a kind of the simplest

mutations which are pyrimidine-to-pyrimidine and purine-to-purine substitutions

Tranversions:the other kind of mutation which are pyrimidine-to-purine and purine-to-pyrimidine substitutions

Point mutations:mutations that alter a single nucleotide

DNA microsatellites: Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites

The Nature of Mutations

transitions transversions

Base change substitutions

Other kinds of mutations(which cause more drastic changes in DNA):

Insertions Deletions Gross rearrangements of

chromosome

These mutations might be caused by insertion by transposon or by aberrant action of cellular recombination processes.

Some Replication Errors Escape Proofreading

The 3’-5’ exonuclease component of replisome only improves the fidelity of DNA replication by a factor of about 100.

But, that’s not enough

The misincorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation.

A mutation may be introduced by misincorporation of a base in the first round of replication.In the second round of replication,the mutation becomes permanently incorporated in the DNA sequence.

Mismatch Repair Removes Errors That Escape Proofreading

Mismatch repair system:a system that increases the accuracy of DNA synthesis by an additional two to three orders of magnitude.

This system faces 2 challenges:(1)rapidly find the mismatches/mispairs

(2) Accurately correct the mismatch

Important parts of mismatch repair system

MutS:a dimer of the mismatch repair protein which detects mismatches

Fuctions of MutS:

1. MutS scans the DNA, recognizes the mismatch from the distortion they cause in the DNA backbone

2.MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself

Functions of MutS

Crystal structure of MutS

Further steps of miamatch repair,we must pay attention to the other two important parts of the mismatch repair system---MutL and MutH

How these three parts interact

MutS-mismatch-containing DNA complex recruits MutL, MutL in turn activates MutH, an enzyme causing an incision or nick on one strand near the site of the mismatch. Nicking is followed by the specific helicase and one of three exonucleases.

Then we talk about :

how does the E.coli mismatch repair system know which of the two mismatched nucleotides to replace?

(Dam)methylation

Dam methylase:the E.coli enzyme that methylases A residues on both strands of the sequence 5`-GATC-3`.

The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.

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a.Replication generates hemimethylated DNA in E.coli.

b.MutH makes incision in unmethylated daughter strand.

Different exonucleases are used to remove single-strand DNA between the nick created by MutH and the mismatch.

Eukaryotic cells

In fact,eukaryotes have multiple MutS-like proteins with different specificities.

MSH proteins MutS homologs

Section 2:DNA Damage

There are mainly three kinds of ways that DNA is damaged:

DNA undergoes damage spontaneously from hydrolysis and deamination

DNA is damaged by Alkylation,Oxidation and Radiation

Mutations are also caused by base analogs and intercalating agents

1st kind, Hydrolysis & Deamination

a.Deamination that Cytosine to Uracil

which explain why DNA contains T instead of U

Answer: If DNA naturally contained

uracil instead of thymine,the deamination of cytosine will create a natrual base which the repair system will not easily recognize.

2nd kind,Alkylation Oxidation and Radiation

DNA is subject to attack from Reactive oxygen species (O2-, H2O2, OH•)

UV(紫外线 ) induces a cyclobutane between adjacent thymines

3rd kind,base analogs & intercalating agents

Base analogs: similar enough to the normal bases to be processed by cells and incorporated into DNA during replication.

But they base pair differently, leading to mistake during replication.

The most mutagenic base anolog is 5-bromouracil,an anolog of thymine.

5-bromouracil,base anolog of thymine,can mispair with guanine

Intercalating agents

溴乙锭

原黄素 丫啶橙

Intercalating agents are flat molecules containing several polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.

Section 3:Repair of DNA Damage

There are two consequences of DNA damage:

Some kinds of damage create impediments to replication or transcription

Other kinds of damage create altered bases that cause mispairing which results a permanent alternation to DNA

Systems that repair damage to DNA

A repair enzyme simply reverses the damage

Excision repair systems,in which damaged nucleotide is not repaired but removed from DNA(more elaborate step),composed of base excision repair and nucleotide excision repair

Systems that repair damage to DNA

Recombinational repair,which is employed when both strands of DNA are damaged,also known as double-strand break repair.(more elaborate)

Translesion DNA synthesis,the last way cells choose

Direct reversal of DNA damage

For example,photoreactivation,which directly reverses pyrimidine dimers

Direct reversal od DNA damage

Another example,methyltransferase(甲基转移酶 ) directly removes the methyl group from the O6-guanine residue

Base excision repair systems

Base excision repair enzymes—glycosylase(糖基化酶 ) recognize and remove damaged bases by a base-flipping mechanism,hydrolyzing the glycosidic bond.

DNA glycosylases are lesion-specific.

1.The AP site is created by the hydrolysis of glycosylase bond.

2.AP endonuclease&exonuclease cut out the 5’ phosphate.

3.DNA polymerase fill in the gap.

The damaged base which is filpped out

The enzyme

The DNA

Nucleotide excision repair systems

What is the difference between the two kinds of excision repair systems?

Also,how does the nucleotide```work? Recognize distortions to the shape

of the DNA double helix Remove a short single-stranded

segment that includes the lesion. DNA polymerase/ligase fill in the

gap.

Once encountering a distortion UvrA exits the complex and UvrB melts the DNA to create a single-strand bubble around the lesion.

Next,UvrB recruits UvrC,and UvrC creates two incisions in different positions on one strand.

Finally,DNA polymerase and ligase fill in the gap.

Recombinational repair

This is the very essencial way that cells repair double-strand breaks in DNA in which both strands of the duplex are broken.

We call it double-strand break(DSB)repair pathway,which retrieve sequence information from sister chromosome.

Translesion DNA synthesis When cells cannot repair certain lesions,there is

a fail-safe mechanism that allows the replication machinery to bypass these sites of damage----translesion synthesis

Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site.

Translesion polymerase is produced by cell in response to the DNA damage

Translesion polymerases are expressed as part of the SOS response pathway.

Crystal structure of a translesion polymerase.

Translesion DNA synthesis in E. coli