Mismatch Repair (MMR) Three roles: Primary function is to correct DNA mismatches generated during DNA replication, thereby preventing mutations from becoming permanent in dividing cells Correction of certain types of DNA damage (spontaneous deamination, oxidation, methylation) Association with sensitivity to alkylating agents Correction of heteroduplex mismatches occurring during homologous recombination

Mismatch Repair (MMR) in E. coli Key components: MutS- sliding clamp ATPase that recognizes mismatch MutL- ATPase that couples mismatch recognition by MutS to down- stream processing steps MutH- hemi-methyl-specific DNA incision-> depends on the lack of DNA methylation newly synthesized strand mismatch binding -> ATP binding & sliding -> incision -> excision -> gap filling -> ligation

MMR components and mechanisms are highly conserved

Models for mismatch recognition in human cells

MMR resolution EXO1 –excision RPA –Involved in all phases of MMR-> binds to nicked heteroduplexes, stimulates excision, protects ssDNA in gap, and facilitates resynthesis perhaps by displacing MutS and MutL HMGB1 –May be involved in DNA unwinding –Can substitute for RPA in in vitro reconstituted system Pol  –Gap filling

MMR deficiency and drug resistance: a paradox Cells that acquire resistance to alkylating agents such as N-methy-N’-nitro-N-nitrosoguanidine (MNNG) often do so by inactivating MMR –Resistance to chemotherapy agents –Many colon cancers are resistant to alkylating agents How does loss of a DNA repair pathway promote resistance to DNA damaging agents? –Disconnect between damage sensing and effector functions

MMR repair signals to checkpoint and apoptotic pathways

Mutations in MMR genes lead to microsatellite instability (MSI) Dynamic expansion and contraction of short sequence repeats Origins of sequence alteration: replication (pol slippage, hairpin formation) recombination (slippage, hairpin formation during resolution) repair (strand slippage during DSB repair) MSI can arise as a result of somatic mutations or germline inherited mutations Germline mutations result in predisposition to cancer, particularly of the colon MSI is used as a diagnostic tool for colon cancers tumors with MSI have different characteristics and outcomes MSI may also have additional deleterious effects by mutating critical genes that contain short repeats

HNPCC—Hereditary Non-Polyposis Colon Cancer Approximately 2-7% of all colon cancers have MSI Dominant inheritance but with incomplete penetrance –Classic tumor suppressor model Cancer General Population Risk HNPCC RisksMean Age of Onset Colon5.5%80%44 years Endometrium2.7%20%-60%46 years Stomach<1%11%-19%56 years Ovary1.6%9%-12%42.5 years Hepatobiliary tract<1%2%-7%Not reported Urinary tract<1%4%-5%~55 years Small bowel<1%1%-4%49 years Brain/central nervous system <1%1%-3%~50 years

Replication of normal undamaged DNA Leading and lagging strand synthesis directed by high fidelity polymerases Fidelity of replication is maintained by: –High nucleotide selectivity –Intrinsic 3’ exonuclease –RPA and PCNA act to suppress deletions or rearrangements arising at repeats

Translesion DNA Synthesis Mechanism for tolerating, rather than repairing DNA damage

Eukaryotes utilize an assortment of DNA polymerases with varying fidelities Note particularly the low fidelity characteristic of the Y family of polymerases— these are particularly active in TLS

Polymerase fidelity is, in largest part, achieved by high nucleotide selectivity generated by tight shape complementarity of base pair binding pockets

TLS involves switching between high and low fidelity polymerases to bypass DNA damage sites

How is TLS controlled? Need to control access of low fidelity polymerases to DNA Plosky and Woodgate Curr. Op. Genetics & Dev. 14:113 (2004) TLS pols tethered to PCNA during normal replication? ubiquitination of PCNA at stalled fork SUMO-Ub switch leads to Pol switch to by-pass damage

DNA double-strand breaks (DSBs) Unlike types of damage we have discussed up until this point, DSBs represent a structural failure in DNA –May occur as a primary defect or secondary to other damage (e.g., consolidation of nearby single stranded gaps –Both exogenous causes (radiation, radiomimetic chemicals) or endogenous (gene rearrangement, meiosis, uncapped telomeres, ROS damage, replication fork collapse) –Most dangerous class of DNA damage to a cell Repaired by one of two different pathways –Non-homologous end-joining (NHEJ) –Homologous recombination (HR)

HRNHEJ Overview: –HR is an extremely accurate mechanism that relies on homology with a sister chromatid to direct DNA synthesis based repair –NHEJ is an error- prone mechanism in which ends are processed to make them compatible and then ligated together

The choice of mechanism for DNA DSB repair Cell cycle regulation: HR requires sister chromatid as a template, thus is more prevalent in S and G2 phase of cell cycle Damage type: Replication fork stalling creates a single double strand end which cannot readily be resolved by simple ligation as in NHEJ

NHEJ core factors Ku70/Ku80 form a heterodimer that acts to protect ends and recruit DNA-PKcs. DNA-PKcs originally identified as a transcription factor plays both a regulatory (via phosphorylation) and structural role. Ligase IV is uniquely used in NHEJ. Has little activity without its co-factor XRCC4.

Ku dimers load onto DNA ends, protects termini and recruits DNA-PKcs DNA-PKcs tethers ends and undergoes phosphorylation and conformational change exposing ends Ends are processed: –direct ligation –polymerase filling/ Artemis digestion –single strand ligation mediated by Cernunnos followed by gap filling Ligation by Ligase IV/XRCC4 complex NHEJ Mechanism

NHEJ is critical for lymphocyte development Mutations in different NHEJ components result in differing types of immunodeficiency: DNA-PK—SCID Ligase IV—LIG4 Syndrome Artemis—RS-Scid Cernunnos—SCID with microcephaly Both B and T cell development depends on the rearrangement of cell surface receptors--a risky strategy that depends upon NHEJ

Lymphocytes incise DNA using the products of the RAG1 and RAG2 genes at sites defined by conserved heptamer/nonamer sequences RAGs are under stringent transcriptional regulation to keep them silenced for all by a narrow window during lymphocyte development

VDJ recombination: role of RAGs

VDJ recombination: end processing

VDJ recombination: resolution

In B cells, NHEJ also takes part in a second rearrangement process: Class switching

Homologous recombination repair of DNA DSBs D loop formation by DNA strand invasion of a Rad51 ssDNA filament Second strand invasion results in Holliday junction formation Resolution via a resolvase or BLM/topoisomerase

Components of HR in eukaryotes

Interstrand crosslinks (ICLs) Important medical significance: many chemotherapeutics cause ICLs (cis-platin, mitomycin C, nitrosourea and derivatives) A single ICL can kill repair-deficient yeast, ~40 can kill repair-deficient mammalian cells. WT yeast can tolerate ~120 ICLs WT human cells can tolerate ~2500 ICLs Induce mutations and rearrangements -> multiple repair factors & pathways working together -> inhibit DNA replication and transcription -> sensitivity to ICL is a hallmark of HR deficiency

ICLs and processing: multiple pathways working together Biological effects are dictated by: cellular uptake metabolic activation types and distribution of adducts -> how diverse? how well are they recognized?

Genetic screens in yeast uncovered ICL-specific repair factors SNM1, PSO2, PSO3, PSO4 -> ICL-specific Relationship to other repair genes? Chanet et al. Mutation Res. 145:145 (1985) wt rad2 pso2rad2 rad52 rad52 Mammalian homologues of some of these genes (SNM1, PSO2) have been isolated and are somehow involved in ICL response

Three classes of ICL repair mechanisms (1)“bacterial-like” A: NER -> HR B: error-prone synthesis -> NER

Three classes of ICL repair mechanisms (2) “DSB initiated” HR A: Two DSBs liberate the damage -> HR B & C: One DSB -> HR

Three classes of ICL repair mechanisms (3) Replication-induced A: HR bypasses ICL at a stalled replication fork B: Bypass of ICL at a stalled fork by HR and translesion synthesis

Summary of yeast ICL repair mechanisms There is no single pathway for ICL repair Factors that affect repair pathway used: -> type and position of ICL -> cell cycle position -> ploidy -> chromatin structure

Kuraoka, I. et al. J. Biol. Chem. 2000;275: Model for repair of an interstrand DNA cross-link S. cerevisiae has multiple mechanisms for ICL repair This represents one proposed model in mammals Note the use of components from different repair pathways, HR and some parts of the NER pathway However, mammals have an entirely unique pathway involved in ICL response....