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Mismatch repair (MMR)Correction of mismatched nucleotides and small loops The mechanism of mismatch repair has been studied most thoroughly in E. coli. Several research groups have re-constituted the repair process from purified proteins. The proteins that initiate the repair process in E. coli are MutS, MutL, and MutH. 1 How do mismatches arise? Most mismatches are due to replication errors. However, mismatches can also be produced by other mechanisms--for example, by deamination of 5-methyl cytosine to produce thymidine (T) improperly paired to G. Regardless of the mechanism by which they are produced, mismatches can always be repaired by the mismatch repair pathway. In cases where the appropriate DNA-N-glycosylase is available, mismatches can also be repaired by the base excision repair pathway. The previous systems recognized DNA damage caused by mutagens. They search for abnormal chemical structures, CPDs, crosslinks. BUT – they can not correct mismatches resulting from replication errors because the mismatched nucleotide is not abnormal in any way – it is simply A,T, C or G inserted in a wrong place. MMR system that corrects replication errors detects not mispaired nucleotides itself, but the absence of base-pairing between the daughter and parental strand. Once it wound the mismatch it will excise it and fill the gap. 2 Important question – repair must be made on the daughter strand because it is in this new synthesized strand that the error has occurred: the parent strand has correct sequence. How does the repair know which strand is which? The answer: In E. coli at this stage: •daughter strand is undermethylated •parent strand has a full complement of methyl groups. Daughter strand is undermethylated E. coli DNA is normally methylated at GATC sequences, but the newly synthesized strand is not immediately methylated since polymerases incorporate adenine, not methyladenine into DNA. The adenines on the daughter strand are methylated by a specific enzyme Dam methyltransferase, only after a lag of several minutes. During this period a new strand contains hemimethylated sequences. 3 Mismatch repair (MMR) Mismatch repair (MMR) in E. coli The replication-error-produced mismatch in the above diagram is indicated by the distorted double helix. 1. MutS protein recognizes such mismatches (true mismatches plus insertions/deletions of up to 4 nucleotides) and binds to them. MutS activates binding of MutL. Scheme by Dr. Huberman 2. Binding of MutL stabilizes the complex. The MutS-MutL complex activates MutH. 3. MutH which is able to distinguish hemimethylated sequences is thus able to distinguish a new strand (presumably incorrect) from the parental (presumably correct) strand. It locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group, as MutH has endonuclease activity. 4 Mismatch repair (MMR) in E. coli 4. Next the segment of daughter strand containing misincorporated base is excised and replaced with correct sequence. Excision is accomplished by cooperation between the UvrD (Helicase II) protein, which unwinds from the nick in the direction of the mismatch, and a single-strand specific exonuclease of appropriate polarity (one of several in E. coli), followed by resynthesis (Polymerase III) and ligation (DNA ligase). MMR in eukaryotes – proteins involved There are several eukaryotic genes that appear to be homologues of the corresponding E. coli MMR genes both in terms of amino acid sequence and in terms of functional similarities. Whereas MutS and MutL function as monomers, the eukaryotic proteins function as heterodimers. heterodimers. Dimers of MutS homologues are responsible for initial recognition of mismatches and small insertions/deletions. Dimers of MutL homologues interact with the resulting complex, as in E. coli. 5 MMR in eukaryotes – proteins involved Two heterodimers of MutS homologues are found in human cells. One of these dimers (MSH2/MSH6) is called hMutSalpha. It preferentially recognizes single base mismatches and small (1-4 base) loops. The second (MSH2/MSH3) is called hMutSbeta and primarily recognizes loops of a similar size range. It is important to note that these specificities are not absolute; MutSalpha and MutSbeta are individually capable of recognizing both single base mismatches and loops of various sizes. MMR in eukaryotes – proteins involved Three MutL homolog dimers are known: One dimer consists of MLH1 and PMS1(yeast)/PMS2(human) and is called hMutLalpha. The second dimer consists of MLH1 and PMS1(human) and is called hMutLbeta. The third dimer consists of MLH1 and MLH3 and has not yet been assigned a name. hMutLalpha can function with hMutSalpha MutSalpha and with hMutSbeta. hMutSbeta The roles of the other two MutL dimers in MMR are not yet well established. 6 Human mismatch repair proteins MMR in eukaryotes – proteins involved At least two nucleases, exonuclease 1 (5' to 3' on dsDNA substrates) and Flap Endonuclease (FEN-1 or DNase IV; Rad27 in S. cerevisiae) cerevisiae) appear to contribute to mismatch repair in eukaryotic cells, just as exonucleases are thought to be important for mismatch repair in prokaryotes. The precise roles of these nucleases have not yet been clarified. 7 MMR in eukaryotes – proteins involved In eukaryotic cells, several standard replication proteins are needed for mismatch repair. The "clamp" protein, PCNA (a cofactor for both polymerases delta and epsilon), is required to stabilize the MutS and MutL heterodimers at mismatch sites on DNA and is also required during the DNA synthesis step of mismatch repair. This DNA synthesis step also requires RPA (the eukaryotic singlestranded DNA-binding protein), Replication factor C (which loads PCNA onto DNA molecules at primer termini) and DNA polymerase delta. Model for mismatch repair Mammalian MMR involves multi-member families of the E. coli prototype factors MutS and MutL. Heterodimers of hMSH2/6 (hMutSalpha) focus on mismatches and single-base loops (stage I in the figure below, upper strand), whereas hMSH2/3 dimers (hMutSbeta) recognize insertion/deletion loops (II, lower strand). 8 Model for mismatch repair Heterodimeric complexes of the hMutL-like proteins hMLH1/hPMS2 (hMutLalpha ) and hMLH1/hPMS1 (hMutLbeta ) interact with MSH complexes and replication factors. Strand discrimination may be based on contact with the nearby replication machinery. Model for mismatch repair A number of proteins are implicated in the excision of the new strand past the mismatch and resynthesis steps, including pol δ/ε , RPA, PCNA, RFC, exonuclease 1, and endonuclease FEN1 (II, III). MMR components also interact functionally with NER and recombination. 9 Model for mismatch repair Recent crystallographic studies have revealed that a MutS dimer detects the structural instability of a heteroduplex by kinking the DNA at the site of the mismatch, which is facilitated when base pairing is affected. Model for mismatch repair However, DNA damage with similar characteristics, such as that caused by alkylating agents and intercalators, may fool MutS, triggering erroneous or futile MMR. Intact MMR thus confers sensitivity, and as several of these agents are used in chemotherapy, tumours may become resistant to them on the basis of selection for defective MMR, so confounding therapeutic strategies 10 Mismatch repair & Hereditary non-polyposis non-polyposis colorectal cancer • HNPCC – 2-10% of all colorectal cancer (this is at least 10 fold higher than the FAP syndrome); autosomal dominant inheritance • A group of 5 similar syndromes (HNPCC1-5) caused by mutations in the mismatch repair genes; most mutations are in MLH1 and MSH2 • Males heterozygous for mutant HNPCC gene have ~90% lifetime risk of developing colorectal cancer; females have ~70% lifetime risk but also have ~40% risk for endometrial cancer Microsatellite Instability (MIN or MSI, or replication error positive or RER+) • microsatellites: repetitive genetic loci (typically 1-5 bases, repeated 15-30 times) • prone to ‘slipping’ during DNA replication → insertions, deletions, normally repaired - mutator phenotype occurs in HNPCC cases • PCR-tested in cancer patients: occurs in 10+% cases of sporadic colorectal cancer •occurs in >90% in tumors from HNPCC family members 11 Mutator Phenotype following loss of mismatch repair • Loss of one allele does not impair mismatch repair • Inactivation of remaining allele (usually allele loss) causes cell to acquire “mutator phenotype” phenotype” • RER+ -replication error positive phenotype • hundreds of errors arise with each round of cell replication and fail to be recognized and repaired – subset of these are likely to activate oncogenes and inactivate tumor suppressors DNA Mismatch Repair Repair of Replication Errors Mechanisms for Insuring Replicative Fidelity 1. Base pairing 2. DNA polymerases - base selection - proofreading 3. Accessory proteins - single strand binding protein 4. Mismatch correction 10-1 to 10-2 10-5 to 10-6 10-7 10-10 12 Eucaryotic homologs of MMR genes Germline mutations occur in the syndrome: Hereditary nonpolyposis colon cancer - HNPCC Approx. 90% of MMR mutations occur in Msh2 and Mlh1 HNPCC accounts for approx. 3% of all colon cancers Mismatch Repair Mutations in Hereditary Nonpolyposis Colon Cancer (HNPCC) • MMR mutations in 70% of families • MLH1 (50%), MSH2 (40%) • Minor role for MSH6, PMS1, PMS2 • Population prevalence 1:2851 (15-74 years) • 18% of colorectal cancers under 45 years • 28% of colorectal cancers under 30 years 13 Functions of MMR Proteins Repair of mismatches and insertion/deletion loops - Msh2, Msh3, Msh6, Mlh1, Pms2, (Pms1, Mlh3) Meiotic recombination - Msh4, Msh5, Mlh1, Pms2, Mlh3 Mitotic recombination - Msh2, Msh3 DNA damage signaling in apoptosis (alkylation damage) - Msh2, Msh6, Mlh1, Pms2 Repair of DNA Interstrand Cross-links - Msh2, Msh3, Mlh1?, Pms2? Interactions in Mammalian MMR Up to about 12 nucleoti des Msh2/Msh6 MutSα (recognizes base-base mismatch and 1bp IDL) Msh2/Msh3 MutSβ (recognizes 2 to approx. 12 bp IDLs) Mlh1/Pms2 MutLα Mlh1/Pms1 MutLβ Mlh1/Mlh3 14 MMR Genes in Colorectal Neoplasia Population incidence MMR deficiency prevalencea MMR gene mutations hMSH2 hMLH1 hPMS2 Nature of Mutations b Truncating Missense HNPCC Sporadic Cancers ~ 1 in 500 > 90% of kindreds c > 70% f 45% f 49% f 6% f 1 in 20 13% e ~ 65% of CRC with MIg 60% h 35% h 5% h 70% f 30% f 55% h 45% h MMR = Mismatch Repair CRC = Colorectal Cancer a As assessed by presence o microsatellite instability (MI). b Based on MMR mutation that could be precisely defined at the nucleotide level. For the purpose of this table, frameshift, nonsense, and splice site mutations as well as large intragenic deletions were considered “truncating.” Three basepair deletions were counted as missense mutations. f Based on 33 mutations in 47 kindreds. In addition, a hPMS1 mutation was identified in a single kindred (Liu et al., 1996) g Based on 15 cases published as of September 1, 1996. h Based on 20 somatic mutations published as of September 1, 1996. Multi-step pathway for development of sporadic colon cancer Genes responsible for HNPCC and FAP are involved in sporadic colon cancer. 15 HNPCC – disease associated with MMR defficiency References: 1. http://www.web-books.com/MoBio/Free/Ch10D.htm 2. M. Esteller et al., DNA methylation patterns in hereditary human cancer mimic sporadic tumorigenesis. (2001) Hum. Mol. Genet. 10, 3001-3007. 3. Lodish et al., Molecular Cell Biology, Freeman and Co. 4. E. Evans and E. Alani, Roles for Mismatch Repair Factors in Regulating Genetic Recombination. (2000) Molecular and Cellular Biology, 20, 7839-7844. 5. B. Vogelstein and K. Kinzler, 1993, Trends Genet. 9:101 16 Literature sources: T.A. Brown. Genomes, John Wiley and Sons,Inc., New-York,p. 330350 (1999). E.Friedberg, G. Walker, W. Siede. DNA repair and mutagenesis, ASM press, Washington DC, 1995 B. Lewin. Genes VII, Oxford University Press. J. Huberman (2001) DNA repair. Roswell Park Cancer Institute. R. Weaver, Molecular Biology, 2002, McGraw Hill Hoeijmakers, J. Genome maintenance mechanisms for preventing cancer. Nature 411, 366-374 (2001). 17