Human mismatch repair has not yet been reconstituted in a defined system, but a number of activities have been implicated in the reaction, including several that are structural and functional homologs of MutS and MutL. Although the nature of the strand signal (equivalent to the methyl-directed signal in E. coli) has not been defined in any eukaryotic organism, heteroduplex DNAs containing a site-specific, strand-specific nick are subject to mismatch-provoked, strand-specific repair by human cell extacts in a reaction that requires ATP, the four dNTPs, and a divalent cation. Since covalently closed circular repair products are recovered at the end of the reaction, DNA ligase presumably seals the strand break that remains after repair DNA synthesis. The general outline of the human mismatch repair pathway is shown in Figure L1.
The mismatch repair (MMR) process is highly conserved throughout evolution. In eukaryotes, the mismatch recognition function is fulfilled by two heterodimeric factors composed of MutS homologs MSH2 and MSH6 (MutSalpha) or MSH2 and MSH3 (MutSbeta). The former recognizes base-base mismatches and small insertion-deletion loops (IDLs) , whereas the latter binds IDLs larger than one extrahelical nucleotide with high efficiency. The MutL function also is conserved in the form of MutLalpha, a heterodimer of MLH1 and PMS1 (hMLH1 and hPMS2 in humans). Interestingly, no MutH homologs have been found to date; this function appears to exist only in gram-negative bacteria. In Streptococcus pneumoniae, mismatch correction is accomplished without methylation direction, and a similar situation must exist in organisms that do not methylate their DNA, such as Saccharomyces cerevisiae and Drosophila melanogaster. In extracts of cultured D. melanogaster and human cells, mismatch-dependent DNA degradation is targeted to either strand of a heteroduplex by pre-existing nicks or gaps, and it has been shown that the repair tracts commence at these strand interruptions. The system appears to lack a mismatch-dependent endonuclease activity, as covalently closed circular substrates are refractory to mismatch repair.
The link of MMR defects with hereditary nonpolyposis colon cancer (HNPCC; see below) has prompted an intensive search for the missing members of the eukaryotic MMR repairosome. Although the DNA helicase and the 3'-5' exonuclease(s) remain to be identified, the system could be shown to involve, in addition to MutSalpha, MutSbeta, and MutLalpha, also the 5'-3' exonuclease EXO1, PCNA, RP-A, and polymerase-delta . The participation of PCNA, which acts as a processivity factor for polymerase-delta, was not surprising. What was surprising, however, was that this homotrimeric sliding clamp appeared to be required not only in the gap-filling reaction, but also in the steps preceding the degradation of the error-containing strand. PCNA is known to be loaded at DNA termini and the MMR process in human cell extracts commences at such sites.
Biochemical studies substantiated the requirement for PCNA in MMR inasmuch as the repair process in vitro could be inhibited by the addition of a p21Cip1/WAF1 peptide containing the PCNA-binding motif, known to block the interaction of proteins with PCNA. Addition of excess PCNA rescued the repair reaction.
PCNA interacts with hMSH3 and hMSH6 both in vitro and in vivo, and the hMSH6-PCNA interaction is required for the correction of base-base mismatches. PCNA appears to interact with other proteins via a conserved motif Qxx[ILM]xx[FH][FY], followed by a nonconserved sequence containing basic amino acids and often also prolines. This motif has to date been found in several human proteins involved in cell cycle control and DNA metabolism, such as the cell cycle regulatory protein p21Cip1/WAF1 (hCDN1), the flap endonuclease FEN1 (hFEN1), the nucleotide excision repair endonuclease XPG (hXPG), DNA methyl transferase I (hMTDM), DNA ligase I (hDNL1), uracil DNA glycosylase (hUNG), and the small subunit of DNA polymerase-delta (hp66). Moreover, a partial PCNA-binding motif was found in the large subunit of replication factor C (RFC1), the catalytic subunit of DNA polymerase-delta (p125), and in the Werner Syndrome gene product.
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Note: A heterodimer of hMSH2 and hMSH6 active in mismatch repair has been designated hMutSalpha. Another heterodimer between hMSH2 and hMSH3 is called hMutSbeta.
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Note: The yPMS1 and yMLH1 yeast proteins interact, and the corresponding human proteins hPMS2 and hMLH1 form a heterodimer designated hMutLalpha that is active in mismatch repair.
Human MutSalpha binds to base-base mismatches and small insertion-deletion heterologies and restores nick-directed repair on both types of substrate to extracts of cell lines that are genetically deficient in MSH2. Human MutSbeta complex binds to insertion-deletion mispairs but displays little if any affinity for base-base mismatches. Furthermore, cell lines genetically defective in the MSH6 subunit of MutSalpha are deficient in base-base mismatch correction but retain partial activity on insertion-deletion mispairs, and this residual activity has been shown to be due to the MutSbeta complex. Thus, MutSalpha participates in base-base and insertion-deletion mismatch repair, but MutSbeta is apparently restricted to processing insertion-deletion heterologies.
A human MutL activity has been isolated by virtue of its ability to restore strand-specific mismatch repair to extracts of one class of hypermutable tumor cell line. This activity, called MutLalpha, is a heterodimer of MLH1 and PMS2. Since cell lines deficient in MutLalpha are defective in repair of base-base and insertion-deletion mismatches, and since the MLH1-PMS2 heterodimer restores repair on both types of heterology, MutLalpha presumably functions with MutSalpha in base-base mismatch repair or with either MutSalpha or MutSbeta in insertion-deletion heterology repair. Defects in either MutSalpha or MutLalpha block mismatch correction at or prior to the excision stage of repair, consistent with function of both activities at an early step in repair.
Certain conditional S. cerevisiae PCNA mutants are hypermutable under permissive conditions, and genetic analysis shows that this increased mutability is due to participation of PCNA in a pathway that also depends on yeast MSH2, MLH1, and PMS1 (human PMS2 is a homolog of yeast PMS1). It has also been demonstrated that yeast PCNA interacts physically with the MutSbeta heterodimer. PCNA, like MutSalpha and MutLalpha, is involved in an early step of mismatch repair.
The repair DNA synthesis step of mismatch repair in human extracts is blocked by aphidicolin which inhibits polymerases alpha, delta, and epsilon. Extracts depleted of polymerase delta, but containing about 5% residual polymerase epsilon and near normal levels of polymerase alpha, are proficient in mismatch-provoked excision but defective in the repair DNA synthesis step. The residual amounts of polymerase alpha and epsilon in the depleted extracts are clearly not sufficient to support the repair DNA synthesis step. However, this defect is alleviated by adding polymerase delta, implicating this polymerase in the reaction.
Little information is available about the proteins involved in the excision pathway of mammalian mismatch repair. Helicase or exonuclease involvement in the biochemical reaction has not been reported. There are, however, hints about the activities that may be involved. Mutations in the exo1 gene of S. pombe, which encodes a 5'-to-3' exonuclease active on duplex DNA, have been implicated genetically in mismatch repair. Mutations in the S. cerevisiae RTH1 gene, which encodes a homolog of the mammalian FEN-1 (a 5'-to-3' exo/endonuclease), also causes an increase in mutability.
More than 20 years ago it was proposed that genetic destabilization would predispose a cell to tumor development. Dramatic support for this idea has appeared during the past several years with the identification of a class of tumor cells that contain frequent mutations in microsatellite repeat sequences like (CA)n or (A)n. The microsatellite instability (MIN+) phenotype is characteristic of virtually all tumors that occur in individuals with hereditary nonpolyposis colon cancer (HNPCC) but is also relatively common in sporadic tumors occurring in a variety of tissues. For example, at least 90% of affected individuals in HNPCC kindreds harbor heterozygotic defects in the genes encoding MLH1, MSH2, or PMS2 with mutations in the PMS2 locus accounting for only a minor fraction of affected families. As might be anticipated from the heterozygotic mode of inheritance of the disease, unaffected cells in HNPCC individuals are typically MIN- and proficient in mismatch repair, whereas the MIN+ phenotype of tumor cells is attributable to inactivation of the wildtype allele of the repair gene in question and loss of mismatch repair capability. Inactivation of the mismatch repair system in MIN+ tumor cells results in a dramatic genetic destabilization. Mutation rates scored either at microsatellite repeat sequences or at the HPRT locus are 100-1,000 times higher in these cell lines than in repair-proficient cells.