Definitions: ★ replication errors ★ spontaneous DNA damage ★ DNA mutations ★ double-strand break (DSB) repair pathway

Questions to be addressed How does the mismatch repair system accurately detect, remove and repair the mismatch resulting from inaccurate replication? What are the environmental factors that cause DNA damage?

How could a DNA damage be converted to DNA mutation? What are the mechanisms to repair a DNA damage? Describes how base excision repair and nucleotide excision repair work? What is translesion DNA synthesis? Why it is important?

The mutability and repair of DNA Replication errors and their repair DNA damage Repair of DNA Damage

Replication errors and their repair Rapair: Mismatch repair Proofreading

The nature of mutations Simple mutations: Transitions(pyrimidine-to-pyrimidine and purine-to-purine) Transversions(pyrimidine-purine and purine-to-pyrimidine) Insertions and deletions (a nucleotide or a small number of nucleotides) ★point mutations: mutations that alter a single nucleotide

Other kinds of mutation: ----cause more drastic changes in DNA Extensive insertions and deletions Gross rearrangements Such changes might be caused by the insertion of a transposon or by the aberrant actions of cellular recombination processes

hotspots—some sites on the chromosome where mutations arise at high frequency while other sites undergoing alterations at a comparatively low frequency about 10-6 to 10-11 per round of DNA. DNA microsatellites —one kind of sequence that is particularly prone to mutation merits special comment , because of its importance in human genetics and disease . They are repeats of simple di-,tri- or tetranucleotide sequences, which are known as DNA microsatellites. (eg.dinucleotide sequence CA)

The replication errors escape proofreading Proofreading improves the fidelity of DNA replication by a factor of about 100.The proofreading exonuclease is not poolproof. If the misincorporated nucleotide is not subsequently detected and replaced, the sequence change will become permanent in the genome.

Mismatch repair removes errors that escape proofreading ★Increase the accuracy of DNA by an additional 2-3 orders of magnitude ★Two challenges: ●scan the genome rapidly ●correct the mismatch accurately ( that is it must recognize the newly synthesized strand) E..coli we take for example

MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone

MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself. The complex of MutS and the mismatch-containing DNA recruits MurL. MutL,in turn, activates MutH, an enzyme that causes an incision or nick on one strand near the site of the mismatch. Necking is followed by the action of a specific helicase(UvrD) and one of three exonucleases.

The helicase unwinds the DNA ,starting from the incision and moving in the direction of the site of the mismatch ,and the exonuclease progressively digests the displaced nucleotide. This action produces a single-strand gap, which is then filled in by DNA polymeraseⅢ and sealed with DNA ligase.

ATP The MutS protein of Escherichia coli MutS is responsible for recognizing and binding to base pair mismatches, and recruits other key proteins (MutH and MutL) required for repair to the mismatch site.

? How does the E..coli mismatch repair system know which of the two mismatched nucleotides to replace？ Dam methylases tags the parental strand by transient hemimethylation and methylates A residues on both strands of the sequence 5’-GATC-3’. MutH protein become activated only when it is contacted by MutL and MutS located at a nearby mismatch. NEXT PAGE

Different exonucleases are used to remove single-stranded DNA between the nick created by MutH and the mismatch. It’s all depending on whether MutH cuts the DNA on the 5’ or the 3’ side of the misincorporated nucleotide. NEXT PAGE

Unmethylated GATC is 5’ of mutation Unmethylated GATC is 3’ of mutation

When it comes to eukaryotic cells MSH == MutS homologs MLH or PMS == MutL homologs Eukaryotes have multiple MutS-like proteins with different specificities.

DNA damage Three reasons for DNA damage hydrolysis(水解) and deamination(去氨基) Alkylation, Oxidation, and Radiation base analogs and intercalating agents

DNA undergoes damage spontaneously from hydrolysis(水解) and deamination(去氨基) This is ironic since the proper structure of the double helix depends on an aqueous environment.

Deamination C-U Depurination ----> an abasic site Deamination of 5-mC---->T

DNA is damaged by Alkylation, Oxidation, and Radiation Often mispqir with thymine G:C –A:T Reactive oxygen species O2-, H2O2, OH• G modification (alkylation & oxidation)

Thymine dimer by ultraviolet light Incapable of base-pairing and cause the DNA polymerse to stop during replication Thymine dimer by ultraviolet light

Clastogenic – ionizing radiation and agents like bleomycin that cause DNA to break are said to be clastogenic.

Mutations are also caused by base analogs and intercalating agents Base analogues

Intercalating agents which cause the deletion or addition of a base pair or a few base pairs

Repair of DNA damage The consequences of damage to DNA Impediments (not permanent) Mispairing(can cause permanent alteration)

Direct reversal of DNA damage Photoreactivation (the enzyme DNA potolyase captures energy from light )

Methyl group removal (a methyltransferase removes the methyl group by transferring it to one of its own cysteine residues)

Base exicision repair enzymes remove damaged bases by a base-flipping mechanism Base excision repair & Nucleotide excision repair

Base excision pathway A glycosylase acts by hydrolyzing the glycosidic bond & then DNA polymerase and DNA ligase restore an intact strand

Structure of a DNA-glycosylase complex DNA glycosylase leision-specific and cells have multiple DNA glycosylases with different specificities

? & The DNA-glycosylase complexs diffuse How do DNA glycosylases detect damaged bases while scanning the genome? Due to the flexibility of DNA ,the damaged base is flipped out so that it projects away from the double helix & The DNA-glycosylase complexs diffuse laterally along the minor groove of the DNA until a specific kind of lesion is detected

If a damaged base is not removed by base excision before DNA replication A fail-safe system

Use the undamaged DNA as a template Nucleotide excision repair enzymes cleave damaged DNA on either side of the lesion ★recognize distortions & ★ a single-stranded gap in the DNA ★ DNA polymerase or ligase fill in the gap. Use the undamaged DNA as a template

Look at the next picture for detail E..coli When it comes to There are four proteins about nucleotide exicision: UvrA, UvrB, UvrC, UvrD Look at the next picture for detail

2 3 1 4

1.UvrA and UvrB scan DNA to identify a distortion 2. UvrA leaves the complex,and UvrB melts DNA locally round the distortion 3. UvrC forms a complex with UvrB and creates nicks to the 5’ side of the lesion 4. DNA helicase UvrD releases the single stranded fragment from the duplex, and DNA Pol I and ligase repair and seal the gap

Transcription coupled DNA repair: nucleotide excision repair system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template

Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA This is accomplished by the double-strand break (DSB) repair pathway

Damage in the DNA template can lead to DSB formation during replication

DSB repair model for homologous recombination

Translesion DNA synthesis enables replication to proceed across DNA damage Occurs when the above repairs are not efficient enough a fail-safe or last resort mechanism, which spares the cell the worse fate of an incompletely replicated chromosome

Translesion DNA synthesis Catlyzed by a specialized class of DMA polymerases that synthesize DNA directily across the site of the damage

Crustal structure of a translesion polymerase

The enzyme is not ‘reading’ sequence information from the template SOS response Translesion synthesis is often highly error-prone

THAT'S ALL