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SPECIAL ARTICLE Molecular Mechanisms Underlying Hereditary Nonpolyposis Colorectal Carcinoma Michelle S. Rhyu* not review nucleotide excision repair and base excision repair, MutS and MutL are bacterial genes that have critical roles in both of which are distinct from mismatch repair in the types of DNA repair and recombination. Mutations in homologues of DNA lesions that they target. Reviews on these subjects can be these genes cause hereditary nonpolyposis colorectal car- found elsewhere (4,12-14). cinoma and are implicated in some sporadic (nonhereditary) colorectal cancers. Molecular functions of these genes have What Is Mismatch Repair? been defined through extensive work in bacteria and yeast. This article reviews and explores molecular events that reThe term "mismatch repair" was initially coined to refer to a quire MutS and MutL, including mismatch repair, homolocellular activity capable of recognizing abnormal base pairs and gous recombination, and gene conversion. The mechanisms correcting the sequence on one strand to retrieve a normal A-T of action of eukaryotic MutS and MutL homologues are comor G-C pairing. This activity was also found to correct stretches pared with those of their prokaryotic counterparts, and the of unpaired bases that result from insertion or deletion of relevance of these mechanisms to tumorigenesis is discussed. nucleotides on one of the two DNA strands. Study of these ac[J Natl Cancer Inst 1996;88:240-51] tivities in Escherichia coli proved that multiple mismatch repair pathways exist (75). Although these pathways are distinguishCellular mechanisms for preserving genetic information were able by their unique substrate specificities and mechanisms of first recognized in the 1960s, when mutants that caused an in- action, two of these pathways, termed "long-patch" and "very crease in mutation rates were isolated in bacteria. At that time, short patch" (VSP) repair, are relevant to the current discussion genes that repair damaged or mismatched DNA were acknow- because of their common reliance on two enzymes, MutS and ledged to be essential for maintaining the integrity of the MutL (75). (The pathways are named for the distinct lengths of genome. As an extension of the multiple-hit theory of cancer, DNA sequence excised on one strand during repair.) This article Loeb (7\2) predicted that mutations in DNA repair genes, by ac- will elaborate on the mechanism of long-patch repair, which is celerating the rate of mutations, would ultimately lead to the more prevalent of the two systems and which has been extumorigenesis. A stream of experiments during the past 2 years tensively studied in E. coli and Streptococcus pneumoniae. For has provided support for Loeb's hypothesis. It is now well ap- clarity, it is noted that bacterial long-patch repair is also known preciated that a number of DNA repair genes, when mutated in as "methyl-directed mismatch repair" in E. coli (75) and as humans, can lead to a predisposition for certain cancers (3-5). "hex"-dependent repair in S. pneumoniae (16). The apparently For example, mutations in human homologues of two bacterial analogous system in eukaryotes has been alternatively termed mismatch repair genes appear to account for an overwhelming "nick-directed mismatch repair" (9) and "MutHLS-like repair" proportion of the tested cases of hereditary nonpolyposis (77). What are the components of the long-patch repair pathway, colorectal carcinoma (HNPCC) (6-8) (Table 1). What is the mismatch repair pathway, and how can it inform and how do they implement DNA repair? Dissection of this sysour understanding of HNPCC? A grasp of molecular tem in E. coli has identified key players and established a mechanisms of action is essential to appreciate how a dysfunc- paradigm for how mismatch repair can work. Subsequent idention in this pathway may lead to cancer. Moreover, under- tification of homologous genes displaying similar functions in standing these mechanisms will help to pave the way to other organisms has cemented the ubiquity of the basic misclassifying cases of HNPCC and to designing therapies. The recent activity in this field has prompted exhaustive reviews of the molecular and clinical aspects of HNPCC (9-7/). This ar•M. S. Rhyu, Ph.D., was a Technology Transfer Fellow at the Journal of the ticle does not attempt to be comprehensive; instead, it presents Cancer Institute, National Cancer Institute, Beihesda, MD, when this an overview of the basic concepts involved in understanding the National article was written. molecular role of mismatch repair in the cell. The article will See "Notes" section following "References." 240 SPECIAL ARTICLE Journal of ihe National Cancer Institute, Vol. 88. No. 5, March 6. 1996 Table 1. HNPCC germline mutations* Kindreds Genetic locus No. % hMSH2 hMLHl hPMSl hPMS2 Otherlocit Total 21 11 1 2 13 48 44 23 2 4 27 100 •Table kindly provided by Dr. B. Vogelstein. fNote that some of these kindreds may belong to one of the loci listed above. Because the tests for mutations have limited sensitivity, some mutations in these loci may have been missed. match repair pathway. While experiments in eukaryotic systems suggest that some important variations from the bacterial model may be used by more complex organisms, experimentation and discovery in bacteria introduced the theory and techniques that continue to inform and instruct the mismatch repair field. Mismatch Repair in Bacteria Beginnings The concept of mismatch repair emerged from the convergence of several independent lines of experimentation: 1) Beginning in the 1950s and through the 1970s, bacterial geneticists identified strains that had a marked increase in the frequency of spontaneous mutations. These were named "mutator" strains and were predicted to be deficient in genes whose normal functions maintain the integrity of the genome (77); 2) in explicating his model for gene conversion, where one allele adopts the sequence of the other allele, Holliday (18) proposed the existence of enzymes capable of repairing mismatches; and 3) investigators studying transformation of 5. pneumoniae by exogenous 5. pneumoniae DNA containing a marker showed that the transforming DNA transiently hybridizes to a homologous site in the endogenous DNA. The "heteroduplex" formed contains mismatches where the marker hybridizes to the endogenous DNA (Fig. 1). It was proposed that repair of these mismatches on the newly introduced strand was responsible for the low efficiency of transformation observed for some markers (79). Mutations at the hex locus appeared to disable the putative repair system, causing all markers to behave as high-efficiency transformers (20). The hex mutants also displayed a "mutator" phenotype (27). These initial experiments are remarkable for the insights they contributed, particularly in light of the relatively primitive experimental tools available at the time. Several reviews (75-77,22) collectively present an impressive picture of how this field unfolded. The high frequency of mutation exhibited by hex mutants (27) gave the first hint that, in addition to correcting mismatches between exogenous and genomic DNA during transformation, the hex locus was required for the more fundamental problem of correcting replication errors. Clear questions arose: When correcting a replication error, how can the cell distinguish between the old and new strand? What signals mediate repair? What proteins are involved? In the course of more than a decade, a number of laboratories utilized various, often clever genetic techniques to answer these questions. In the interest of con- Single Strand of Transforming DNA with mutation Replication and Recombination Low Efficiency Transforming Marker (with repair) High Efficiency Transforming Marker (no repair) Cell "B" Division / Fig. 1. Streptococcus pneumoniae transformation. Figure depicts two alternative outcomes after introduction of a single strand of homologous DNA containing a mutation, or marker (blue dotted fragment). Markers displaying high efficiency of transformation (on left in diagram) incorporate the single strand containing the marker into the chromosome during the subsequent replication (newly replicated strand is in red), yielding _a cell (labeled cell "B") with a heteroduplex DNA. Division of cell B generates a cell containing the sequence of the original bacterial DNA and a cell homozygous for the marker (newly synthesized strand shown in green). Markers displaying low-efficiency transformation are not propagated because of their susceptibility to DNA repair, which displaces the transforming DNA with newly replicated DNA (on right in diagram). \ Displays mutation Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 SPECIAL ARTICLE 241 cision, all of these experiments cannot be explained in depth. Instead, one valuable biochemical technique, the in vitro mismatch repair assay (23,24), will be explained. This assay both validated the conclusions of preceding genetic experiments and established the means to isolate proteins in the mismatch repair pathway. Hind III site 51 31 AAGCTTTCGAG TTCGAQAGCTC In Vitro Mismatch Repair The power of the cell-free assay rests in the manipulability of its constituent parts, the substrate and the extract. The substrate is a double-stranded, circular DNA molecule that contains a mismatched base pair whose repair can be easily evaluated (23). Fig. 2, A, shows an example of such a substrate, where the G-T mispair lies within an imperfect cleavage site for two restriction endonucleases (25). If the mispaired G (on the bottom strand in Fig. 2, A) is corrected into an A, the resulting A-T pair completes the recognition site for the restriction endonuclease, Hindlll; if the T (on the top strand) is corrected into a C, the recognition site for the Xho I endonuclease is formed; if neither base is altered, neither endonuclease can cleave at this site. This type of facile test for evaluating the repair product made it possible to discern the exact substrate features required for repair. For example, addressing the question of how the mismatch repair system might distinguish the old DNA strand from the new strand, containing replication errors, Wagner and Meselson (26) proposed that, in E. coli, the two strands of DNA may be discerned by their methylation states. They (26) noted that normal DNA methylation on A residues within d(GATC) sequences succeeds replication with some delay, causing the transient appearance of "hemimethylated" DNA, where only the old strand exhibits the methyl-group modification (27). Multiple genetic experiments assessing repair activity in vivo supported this theory (1522); in vitro repair of a heteroduplex comprised of one methylated strand and one unmethylated strand confirmed the hypothesis, providing molecular evidence that the unmethylated strand is consistently repaired, using the methylated strand as a template (23). If neither strand is methylated, the determination of which strand to repair becomes random, causing half of the repair events to incorporate the mutation. Using varied substrates, it was also confirmed that of the eight incorrect pairings, A-G, A-C, G-T, C-T, A-A, T-T, G-G, and C-C, only C-C mismatches were refractory to repair by this pathway (15). The other component of the in vitro assay is a cell-free bacterial extract that retains mismatch repair ability. The success of this assay demonstrated that the repair reaction could be reproduced independently of the cell and raised the possibility of reconstituting repair activity using purified protein components. The preceding decades of genetic insights had already identified the mutator genes MutH, MutL, MutS, and UvrD (also known as MutU) as genes required for mismatch repair (28-30); the next obvious step was to purify the proteins encoded by these genes. When extracts were made from each of these mutant strains, mismatch repair activity was lost, but activity could be restored using any combination of the mutant extracts. The ability to recover activity by supplying the missing protein exogenously provided a means to purify each protein: for example, once the MutS gene was identified and cloned into a vector that allowed overexpression of MutS, extracts were made 242 SPECIAL ARTICLE B T-+ C correction makes substrate for Xhol G-»-A correction makes substrate for Hind III 5'CTCGAG QAGCTC 51 51 AAGCTT TTCGAA 5' Extract Fractionate • MutS •[cellular *J proteins Assay for Mismatch Repair Activity With MutS" Extracts MutS + MutS' Extract MMR Competent Fig. 2. Cell-free mismatch repair. A) The substrate. A prototypical substrate for the in vitro assay is a plasmid that contains a mismatched base pair. (Mismatches are depicted here and in subsequent figures as a notch in the DNA.) In this example, the G—T mismatch (in bold) lies within imperfect endonuclease recognition sites for two restriction endonucleases, Hindlll and Xho I This context for the mismatch allows easy detection of the in vitro mismatch repair event. Correction of the T on the top strand into a C restores the Xho I restriction site, whereas correction of the G on the bottom strand to A creates the Hindlll site. The substrate can also be manipulated to test the dependence of repair on other factors, such as DNA strand methylation. (Here one strand is shown methylated.) B) Purification of repair proteins by complementation. The repair protein (MutS in this example) may be isolated by virtue of its unique ability to restore mismatch repair (MMR) activity to an extract defective in that protein (here, MutS"). Extracts made from cells overexpressing MutS" protein (darkened circles) are fractionated biochemically, separating MutS from other cellular proteins (depicted as triangles, squares, and diamonds). The fraction including the purified protein can be identified by its ability to complement the MutS extract. Successive rounds of fractionation and complementation will yield progressively more purified isolates of the protein. with bacteria transformed with this A/wfS-expressing plasmid. These extracts were biochemically fractionated, and the fraction containing MutS was identified through its ability to restore mismatch repair activity to extracts made from /WwfS-deficient cells (Fig. 2, B). By monitoring the protein's activity through multiple purification steps, near-homogeneous preparations of MutS could be isolated and used for biochemical analysis. MutH, MutL, and MutS were all purified in this manner (31-33). UvrD (MutU) had previously been identified as helicase II, a protein that assists at unwinding the double helix (34,35). UlJournal of the National Cancer Institute, Vol. 88, No. 5, March 6. 1996 timately, an in vitro system comprising purified protein components was shown to support the mismatch repair reaction (36). Through the use of cell-free mismatch repair assays, it was determined that bacterial methyl-directed mismatch repair requires 10 proteins acting in the following three steps (Fig. 3): 1) Recognition of the mismatched DNA—MutS protein binds the mismatch (32), and MutH (an endonuclease) binds a nearby (within 2 kilobases) hemimethylated GATC sequence that can be either 3' or 5' to the mismatch (31,37^8). A homodimer of MutL forms a complex with MutS and MutH (59). This binding potentiates the endonuclease activity of MutH, which cleaves the DNA at the d(GATC) site on the unmethylated strand. This reaction requires adenosine triphosphate (ATP) (31,40). 1. CH 3 A 5' 3 3' ~5 V 5 SUM"3 3' " 3' ™—Nicking Site Nick 2. Excision A 5' V exonuclease VII. RecJ. ^(exonuclease 1). helicase CH3 A 5' 31 5' 3' 1 Single strand 1 binding protein 3. Resynthesis CH3 — 3' 5' 31 y^jK^K^J1 DNA polymerase III, I ligase CH3 — 3' 5' 3' 5 Key A MutS 0 Why Do Repair Mechanisms Exist? Replication Errors Recognition 5' 3' 2) Excision of the newly synthesized strand—An exonuclease removes a segment of the incised strand extending from the nick at the d(GATC) site to just beyond the mismatch (38). If the nick is 5' to the mismatch, the 5' to 3' exonuclease activity of Rec J or exonuclease VII is required for this reaction; if the nick is 3' to the mismatch, the 3' to 5' exonuclease activity of exonuclease I is utilized (37). Excision of the nicked strand is predicted to require the DNA-unwinding activity of helicase II (MutU) (55). 3) Replacement of the excised DNA and ligation—The unpaired DNA is stabilized with single-strand binding (ssb) protein, as DNA polymerase III resynthesizes the complementary strand (56), correcting the mismatch. Ligase is required to covalently link the repaired portion to the pre-existing sequence (36). MutL % MutH 0 0 ssb Fig. 3. Bacterial methyl-directed mismatch repair. DNA is depicted as double, black lines. Methylation of d(GATC) on the top strand indicates this is the older DNA strand. Step I: The mismatch is bound by MutS (large, pink oval), the hemimethylated d(GATC) is bound by MutH (in green), and MutS and MutH form a complex via interaction with a homodimer of MutL (small, purple ovals). This interaction causes MutH to nick the hemimethylated d(GATC) on the unmethylated strand. Step 2: Exonucleases and helicase excise the portion of the unmethylated strand from the nick to beyond the mismatch. Step 3: Singlestrand binding (ssb) protein (yellow circles) stabilizes the unpaired DNA as DNA polymerase III resynthesizes the complementary DNA (dotted red line). Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 At first glance, it may seem untenable that the polymerase that copies chromosomal DNA is imperfect. After all, the primary imperative of an organism should be the faithful replication of its genetic material. In fact, the accuracy of the polymerase is challenged by numerous factors (41), two of which are particularly relevant to this discussion: 1) Some nontypical base pairs, though not ideal, are energetically allowable and may be formed at a low frequency (42,43) (Fig. 4, A); and 2) stretches of highly repetitive DNA sequences may cause the polymerase to lose its place and "slip" on the template during replication. If the polymerase slips forward on the template, the newly synthesized strand will contain fewer nucleotides than the template, whereas slipping back to an already replicated nucleotide causes the insertion of extra bases in the new strand (Fig. 4, B). In fact, the error rate of about 1 in 109 to 1 in 1010 base pairs measured for bacterial DNA (17,44,45) replication reflects the corrective influence of two mechanisms that "proofread" the errors of the polymerase. The first mechanism is a 3' to 5' exonuclease activity, which associates with the polymerase and corrects mispairs as they occur on the newly synthesized strand, giving the polymerase a second chance to make a suitable base pair (46,47). But this proofreading mechanism is also imperfect. Mismatched or unmatched base pairs that elude the detection of the exonuclease are generally corrected by the mismatch repair system, which accounts for another 100-fold to 1000-fold increase in the accuracy of replication (17). It is notable that wild-type cells with repair systems intact still exhibit an error rate of 10"10. Why is replication with repair not perfect? A commonly held view, consistent with the paradigm of evolution, suggests that a low rate of mutation (which allows for alterations in the genome) may confer the opportunity to acquire "favorable mutations," which bestow a selective advantage over an organism with a static genome. Recombination Genetic studies in E. coli and yeast showed that repair mutants display an elevated frequency of recombination, inSPECIAL ARTICLE 243 Watson-Crick base pairs CH, O H—N v cr o—H-H' some abnormal pairings cr ct B T T T T T T T T T A A A A A A A A A A A A A A A A A A A A A A A A A A A Fig. 4. A) Standard WatsonCrick base pairs and some energetically possible base pairs. B) Slippage during replication of repetitive sequence. DNA polymerase is shown replicating a DNA strand containing multiple adenine (A) residues in tandem. Repetitive sequence may cause the polymerase to "slip," in this case causing four thymine (T) residues to become unpaired. The replication event diagrammed here would result in four extra T residues on the new DNA strand, if not for the corrective effect of mismatch repair genes. In a similar way, polymerase could slip forward on the template, leaving unpaired A residues and resulting in a new DNA strand containing fewer residues than the template. A T T T T T T T T T T T T T A A A A A A A A A A A A A A A A A A dicating a wild-type role in suppressing recombination events (48,49). Specifically, mismatch repair appears to affect at least two types of genetic recombination, where the formation of a heteroduplex stretch of DNA is an intermediate step in the recombination event. A simplified model of a homologous recombination is diagrammed in Fig. 5, A. Nicks in one strand of a chromosome can initiate exchange of a single strand between two chromosomes, forming an intermediate termed the "Holliday structure," named after the scientist who first proposed it (18). This branched structure is dynamic and may "migrate" along the DNA, determining the extent of singlestrand exchange. Eventually, endonucleases and ligase resolve the Holliday structure in one of two ways: Cleavage and ligation of the "inside strands" depicted in the diagram give rise to a short region of single-strand exchange; but cleavage and ligation 244 SPECIAL ARTICLE A of the "outside strands" result in homologous recombination (50-52). (In three dimensions, the alternative cleavage sites are structurally equivalent.) This type of gene rearrangement ordinarily occurs between nearly identical tracts of DNA sequence. For example, homologous recombination between alleles of the same gene during meiosis in early development of the sperm and egg is crucial to creating genetic diversity. Early investigators (55) determined that as little as 10%-20% divergence between sequences was sufficient to prevent homologous recombination in bacteria. It was subsequently shown that the mismatch repair genes MutH, MutL, and MutS are necessary to implement this restriction. In bacterial strains mutant for any of these genes, the frequency of recombination between similar but highly divergent sequences increases dramatically, while homologous recombination rates are unaffected (54J>5). By allowJournal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 B A i i i i i i i i i 1 I 1 i i I i 1 i i i i i i i i i I I 1 | I I 1 1 1 1 V) 1 1 1 | * " a (M,___ j i i I I 1 l i l t i i i i i i i i i 1 I i i i i i I i i i I I | i i 1 i 1 I 1 1 i i i i i i i i i i 1 1 i i i i i i i i i I I I | \l i I \\ Replication and Meiosis l i I I 1 1 1 1 1 1 | I i 1 1 11 A A 1 I 1 I I 1 1 1 1 1 A A A J ^ A ) ., J nick i i i i i i i i i 1 1 i i i i i i > i i I i Holiiday r V r StructureLA ^> [ I i I i i I I I I I I I i i i i i i I t r 1 I 1 1 1 11 | 1 i 1 1 I 1 | A A A A A A 1 l l 1 a A A r)<ff a A a a a a a a a normal 4A4a gene conversion 6A;2a PMS 5A:3a J__L_L_L I 1 1 | | Branch migration LJ 1 1 t | X i i i i i i i i i I I I JL-i 1 1 | T A HD x ' ' i i i i i i i i i i i i I / \ Y HD * i i i—i— 1 i i XR I 1 i * 1 1 1 , i HD i it HD XR Homologous recombination J Fig. 5. A) One type of homologous recombination event. The two parental chromosomes are depicted in red and black. A nick in the red chromosome allows it to invade its chromosome pair. The displaced strand of the black chromosome may also become nicked, allowing an exchange of DNA strands and forming the Holiiday structure. Extensive homology between the chromosome pairs allows movement of the Holiiday structure along the DNA, causing lengthy exchanges of DNA strands and consequent regions of heteroduplex DNA (HD). The Holiiday structure may be resolved through nicks and ligation either at the sites marked by X or at the sites marked by Y. Nicking and ligation at X leave regions of heteroduplex but no chromosome exchange, whereas resolution at the Y sites causes chromosome arms to be exchanged, resulting in homologous recombination. (XR represents exchanged regions.) B) Gene conversion and postmeiotic segregation (PMS). Rectangle at top represents a diploid yeast cell containing the paternal (P, in black) and maternal (M, in red) alleles of the "A" locus. (Each line represents a single strand of DNA; a pair of lines depicts one chromosome.) After replication and meiosis, the chromosomes are duplicated, and one allele (two DNA strands) is distributed to each haploid spore. Each column shows a tetrad of four spores from one meiosis. Newly synthesized DNA strands are shown in blue and green. The first column shows the products of a normal meiosis, resulting in four paternal and four maternal DNA strands (4A:4a). The second column shows the effects of a gene conversion event, where a segment of the paternal DNA displaces the maternal information, resulting in a 6A:2a distribution. The third column shows the observed 5A:3a DNA strand distribution for PMS mutants. The PMS pattern represents an incomplete gene conversion event, where the paternal DNA segment displaces the maternal segment on one strand, but mismatch repair fails to repair the complementary strand to yield the 6A:2a pattern exhibited in gene conversion. ing rearrangement between similar sequences within a genome, these aberrant recombination events, termed "homeologous recombination," could potentially cause widespread genomic rearrangement and, eventually, cell lethality (56). The precise action of the mismatch repair genes in preventing homeologous recombination is unknown, but an in vitro study (57) suggested that MutS and MutL inhibit the branch migration step of the reaction. Comparable experiments in Sacchciromyces cerevisiae yielded similar results, demonstrating that three MutS homologues, yMSHI, yMSH2, and yMSH3, may also contribute to suppressing homeologous recombination in yeast, a eukaryotic ments of DNA, gene conversion events are characterized by the unidirectional replacement of sequence on one chromosome with sequence from the other (Fig. 5, B). These events have been effectively dissected by studying meiotic gene conversion in S. cerevisiae. In this system, the four haploid cells (spores) descendant from the meiosis of a single cell can be analyzed for their inheritance of specific markers. Ordinarily, the parent cell replicates and distributes its DNA during meiosis to generate two spores containing the paternally derived allele and two spores containing the maternally derived allele (a 4:4 DNA strand inheritance pattern). When gene conversion occurs, a 2:6 or 6:2 pattern is observed, such that either the maternal or paternal allele is overrepresented among the four spores. Postmeiotic segregation (PMS) mutants significantly reduce the frequency of meiotic gene conversion and display an alter- system (52J8). A second type of recombination event affected by the mismatch repair genes is gene conversion. In contrast to homologous recombination, where two chromosomes exchange segJournal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 SPECIAL ARTICLE 245 native inheritance pattern, 5:3 or 3:5, where one spore contains one DNA strand of paternal origin and one of maternal origin (Fig. 5, B) (59). This PMS phenotype suggested that the complete gene conversion is prevented by a failure to repair mismatches in a heteroduplex intermediate; direct examination of heteroduplexes in these mutants confirms a role for mismatch repair in gene conversion (60-62). Given that gene conversion events are often accompanied by homologous recombination events, it is reasonable to imagine that repair of mismatches in the heteroduplex regions (designated in Fig. 5, A) will sometimes lead to gene conversion. Molecular characterization has revealed that PMS-1, a mutant that displays PMS, is a MutL homologue (63) and that yMSH2 and yMLHl likewise have a strong PMS phenotype (52,64-66). Although exact mechanisms are still unclear, one conclusion is evident: Homologues of two long-patch repair genes, MutS and MutL, appear to have important roles in the fidelity of recombination. Some Other Roles Attempts here and generally in the literature to present clear molecular models belie the probability that the identified genes participate in multiple, distinct repair pathways about which we currently know very little (67-69). Mismatch repair pathways are known to recognize aberrant base pairings caused by some postreplicative mechanisms. For example, the normally occurring modified base, 5-methylcytosine, can undergo spontaneous deamination to form thymine (70-72). In such an event, a G-C base pair mutates to G-T. The very short patch repair system, which relies on MutS and MutL, specifically recognizes this type of G-T mispair and restores it to G-C (73,74). MutS and MutL also appear to detect unnatural base methylations caused by alkylating agents. In this case, however, the aberrant base pair is not replaced. Instead, the action of MutS and MutL on these lesions leads to cell cycle arrest in G2 and eventual lethality (5,75). Cells mutated for MutS and MutL fail to initiate G2 arrest and display a tolerance to alkylating mutagens (5,68,75-77). The participation of mismatch repair in these various pathways indicates that this system is utilized as a general sensor of mismatched bases created in multiple ways. Mismatch Repair Genes and HNPCC Compared with the thorough genetic and biochemical characterization of bacterial methyl-directed mismatch repair, understanding of mismatch repair in eukaryotic systems was scant in 1990. During the late 1980s and early 1990s, mismatch repair systems were quietly studied in yeast, Drosophila, Xenopus, and mammalian cell lines. Findings in these systems suggested that fundamental aspects of long-patch repair are conserved in eukaryotes (61,78-82), but these studies groped to understand the wider physiologic relevance of this pathway. All this was changed by the revelation that HNPCC reflects problems in mismatch repair. These developments have heightened interest in the field and produced valuable reagents for studying the molecular mechanisms of eukaryotic mismatch repair. A discussion of these molecular mechanisms necessitates an introduction to HNPCC and its connection to the mismatch repair pathway. While the following summary highlights key 246 SPECIAL ARTICLE advances in the field, I strongly urge the interested reader to consult a recent review by Marra and Boland (JO) for more thorough coverage of HNPCC and a description of the drama of how this disease was linked to defects in mismatch repair. HNPCC, also known as Lynch syndrome (83,84), refers to a subset of colorectal carcinomas, characterized by (a) early age at onset, (b) occurrence of cancer preferentially in the proximal colon, and (c) higher incidence of cancers at noncolonic sites (particularly in the endometrium, but also in the stomach and ovary) among family members. This disparate collection of diseases was suspected to have a genetic basis when autosomal dominant inheritance was demonstrated (85). Still, in the absence of molecular markers such as a restriction fragment length polymorphism (RFLP) linked to the disease, the role of genes in HNPCC remained in question. The detection of "microsatellite instability" in colorectal tumors (86-88) provided the key to unlocking the long-intractable mystery of the molecular cause of HNPCC. Microsatellite sequences, which are dispersed throughout the genome, are stable stretches of DNA composed of repetitive sequences that do not encode proteins. In tumor tissues from HNPCC patients, these sequences were found to consistently contain insertions and deletions (88). Similar alterations were found in some instances of nonhereditary (sporadic) colorectal cancers (86,87). Correlation of microsatellite instability with HNPCC catapulted progress in this field by begetting two distinct approaches to identifying HNPCC genes. Peltomaki et al. (6) used a battery of microsatellite markers to conduct linkage studies, which localized the HNPCC gene to a small region on chromosome 2. Through classical human genetics methods, this mapping information eventually led to cloning of the HNPCC gene at position 2p (89). Meanwhile, some researchers working in bacterial and yeast mismatch repair recognized that microsatellite instability could reflect a defect in a human mismatch repair pathway. In September 1993, Strand et al. (90) first published this proposal, showing that, when yeast strains are mutant for homologues of the bacterial mismatch repair genes MutS or MutL, nucleotides are aberrantly inserted or deleted during replication of a stretch of tandem repeats of poly(dG-dT). This demonstration mimicked the microsatellite instability phenotype seen in colorectal tumors and suggested that this phenotype is the result of polymerase slipping (diagrammed in Fig. 4, B). This rationale led Fishel et al. (91) to clone a human MutS gene (hMSH2, or human MutS homologue) by methods originally used to isolate the yeast MSH1 and MSH2 genes (92). This cloning-by-homology approach yielded the first evidence that HNPCC was caused by a defective mismatch repair pathway. The culpability of the MutS homologue (hMSH2) was corroborated when, 2 weeks after the publication of the article by Fishel et al. (97), Leach et al. (89) reported the cloning of the same gene using traditional genetic methods. Leach et al. (89) also provided convincing evidence of protein-altering hMSH2 mutations isolated from DNA of HNPCC patients. Furthermore, in collaboration with the Modrich laboratory, whose expertise lay in mismatch repair mechanisms, this group (95) produced an accompanying article showing that extracts from tumor cells exhibiting microsatellite instability were indeed defective in an in vitro mismatch repair Journal of the National Cancer Institute, Vol. 88, No. 5, March 6. 19% assay. The in vitro assay satisfied the prediction that hMSH2 has a function similar to that of its bacterial homologue. Palombo et al. (94) subsequently showed that a G-T mismatch binding protein, which they were characterizing, was hMSH2. Within a few frenetic months, it was thus proven that HNPCC and its characteristic microsatellite instability are the result of a malfunction in the mismatch repair pathway. Since these initial revelations, several additional human mismatch repair genes (hMLHl and hPMSl through hPMSS) have been cloned by virtue of homology to MutL and yeast MutL homologues (95-99). Of these, hMLHl, which is located on chromosome 3p, has been confirmed as another major locus for HNPCC (7,8$5). Although HNPCC patients with germline defects in hPMSl (one patient) and hPMS2 (two patients) have also been identified, mutations at these loci are less prevalent than mutations in either hMSH2 or hMLHl in patients tested to date (8$6). Another MutS homologue, GTBP (G-T binding protein), has been identified through biochemical characterization (100,101) and will be discussed below. Equipped with the connection to HNPCC and other colorectal cancers, investigators have begun to understand how eukaryotic mismatch repair works. Their experimental approach has recapitulated the basic strategies set forth in the bacterial experiments: 1) use of heteroduplex substrates to assess repair activity in cells and extracts, 2) in vivo and in vitro assays for loss of repair activity in specific mutants, and 3) isolation and biochemical characterization of proteins essential for repair. These experiments indicate that, for several steps of the bacterial long-patch repair paradigm, eukaryotic repair mechanisms possess some familiar features as well as provocative differences. B hMSH2 GTBP hMLHl ,hMSH2 hPMS2 GTBP I excision excisi from site of nick nicl to mismatch J\. I Polymerization Mechanisms of Mismatch Repair in Eukaryotes Nick-Directed Repair Eukaryotic cells lack d(GATC) methylation. Accordingly, no eukaryotic homologue to the d(GATC) endonuclease, MutH, has been identified. This method of strand distinction must not be used; yet, some method of distinguishing the strands presumably exists. Despite some proposals (9,102), no precise eukaryotic mechanism for strand distinction has been proven. Any mechanism that discerns the old DNA strand from the new strand in order to preserve the sequence on the old strand is predicted to include preferential cleavage of the newly synthesized strand: In human cell extracts, artificially constructed, circular heteroduplex substrates containing a nick in one strand are consistently corrected on the incised strand (80,81), whereas these extracts repair substrates without any nicks with no strand preference (81). This repair activity requires hMSH2 (103), hMLHl (104), hPMS2 (105), and GTBP (100); extracts made from cells mutant for any of these genes lack nick-directed repair activity. Although this observation does not prove that a strand-specific nick is used in vivo, it demonstrates that eukaryotic homologues of bacterial mismatch repair proteins can recognize and use a nick to carry out mismatch repair. Considering the mechanism of DNA replication, it is plausible that the repair machinery on the lagging strand is directed to the newly synthesized DNA strand by its temporary nicks immediately following replication (Fig. 6, A). However, if nicks direct Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 Fig. 6. Eukaryotic nick-directed mismatch repair. A) A replication fork, with purple arrows representing newly synthesized DNA and its direction of synthesis. The region demarcated by the dotted lines is elaborated in B-E. B) hMSH2 and GTBP bind the mismatch as a heterodimer. C) hMLHl and hPMS2 bind the hMSH2-GTBP plus heteroduplex DNA complex. D) The nicked strand is excised from the point of the nick to beyond the mismatch. E) Polymerization and ligation presumably restore the excised strand, correcting the mismatch (restored DNA shown as dotted red line). repair on the leading strand, the repair event may require introduction of additional nicks on this strand. Mismatch Recognition Just as with the bacterial mismatch repair pathway, different heteroduplex substrates have been used in human cell extracts to ascertain the efficiencies with which different mismatched base pairs are corrected by the nick-directed repair pathway (80,106). Like the bacterial system, nick-directed repair in human cell extracts can repair small nucleotide insertions and/or deletions caused by polymerase slippage (80,81). But in addition to the seven mismatches corrected by the bacterial pathway, human extracts can also correct the C-C mispair (79,80). The reason for this difference is unclear. The overall similarity of mismatch specificity nonetheless supports the view that the nick-directed SPECIAL ARTICLE 247 system is analogous to the bacterial methyl-directed mismatch repair system. Despite general similarities, the specifics of mismatch recognition differ between bacterial and human cells. While the MutS protein alone binds the mismatch in E. coli (32,107), hMSH2 in human cell extracts binds mismatched or unmatched base pairs with a partner, GTBP (100,101) (Fig. 6, B). Two groups independently determined that GTBP forms heterodimers with hMSH2. Drummond et al. (100) showed that extracts made from cells mutant for hMSH2 are deficient in repair. Using HeLa cell extracts, they purified an activity capable of rescuing the hMSH2 defect and found that this fraction contained a complex of hMSH2 and a 16O-kd protein, which, upon protein sequencing, was found to be GTBP. They named the heterodimer "hMutSa." Palombo et al. (101) arrived at the same conclusion through cloning the genes for the 100-kd and 160-kd proteins that were isolated on the basis of G-T mismatch binding activity and studying biochemical interactions between the recombinant proteins (94,101). Although hMSH2 and yMSH2 can bind to mispaired base pairs without GTBP (94,108), Palombo et al. (101) showed that strong binding to a G-T mismatch may require both proteins. GTBP itself has homology to bacterial MutS (101). Thus, mismatches appear to be recognized and bound by a heterodimer consisting of two distinct MutS-like proteins. It is not yet understood why eukaryotic cells require a heterodimer for mismatch recognition, when bacteria accomplish the task with one protein. Whatever the explanation, one thing is clear: GTBP and hMSH2 do not have identical functions. Cells mutant in hMSH2 display a broader range of defects than G7'fl/'-deficient cells (100,109), indicating that hMSH2 functions without GTBP for certain repair reactions. For example, GTBP mutant cells have a reduced but notable ability to correct stretches of two, three, or four unmatched bases, while hMSH2 mutant cells lack this ability (100). It is tempting to speculate that hMSH2 forms heterodimers with other MutS-like proteins to assert its GrBP-independent functions. The mutant phenotype of yMSH3 suggests that this yeast gene is preferentially used to correct two base-pair insertions and deletions without influencing nucleotide mismatches or single unmatched pairs (110). A human homologue of yMSH3 would be a strong candidate for another hMSH2-binding partner. After Mismatch Recognition: Binding of MutL Bacteria] MutL has been shown to bind a MutS plus heteroduplex DNA complex as a homodimer (55). While no biological function has yet been attributed to MutL, defects in each of three MutL homologues have been associated with HNPCC patients (95-97), confirming the importance of this gene family in mismatch repair. The human homologues were named on the basis of their extent of homology to the previously identified yeast MutL-like proteins, yMLHl and yPMSl. The human genes are hMLHl, hPMSl, and hPMS2 [which is more homologous to yPMSl than is hPMSl (96)]. Like their prokaryotic counterpart, eukaryotic MutL homologues appear to bind their substrate as dimers (Fig. 6, C). Using protein affinity chromatography, Prolla et al. (66) first demonstrated the physical interaction between yMLHl and yPMSl. Li and Modrich (104) verified the function of this heterodimer in vivo when they 248 SPECIAL ARTICLE determined that the purified activity that restores mismatch repair to hMLHl-deficient extracts is made up of hMLHl/hPMS2 heterodimers. The fact that these MutL homologues form functional heterodimers, combined with evidence that a mutation in hPMSl is also associated with HNPCC, suggests that hPMSl may form a heterodimer with a different MutL homologue. This putative partner may be among the family of six MutL-like genes (hPMS3-hPMS8) located near hPMS2 on chromosome 7. Little is known about these genes, but the sequences so far identified are intriguing in that they predict products resembling truncated forms of MutL-Wkt proteins (98£9). The multiplicity of these MutL homologues and their potential capacity for heterodimerization have led to proposals that each homologue may carry out a specialized function or that the homologues may be expressed in a developmental or tissue-specific pattern (104). Other Downstream Genes Fewer details are known about the later steps in nick-directed repair, but the mechanisms used in bacterial methyl-directed repair may, as expected, be roughly preserved. Like bacteria, human cell extracts possess the "bidirectional" ability to excise the nicked strand from a position either 3' to or 5' to the mismatch (111). Although hMSH2, hMLHl, hPMSl, and hPMS2 account for a large proportion of the HNPCC cases studied to date, a substantial number of HNPCC cases appear to be normal at these loci (8) (see Table 1). Hence, the discovery of other repair loci, representing other essential proteins in this pathway, is anticipated. Mismatch Repair Mechanisms and Cancer How Do Mismatch Repair Defects Cause Tumors? The steps that lead from MutS and MutL defects to HNPCC remain largely unknown. However, it appears that a defect in mismatch repair alone is not sufficient to induce tumorigenesis. Parsons et al. (112) made the unexpected discovery that some HNPCC patients have non-neoplastic lymphoblast cells that are, nonetheless, deficient in mismatch repair. Although colorectal cancers were detected in these patients, their incidence of tumors at noncolonic sites did not approach the expected frequency, considering the elevated rate of mutation at these sites. The mutagenic phenotype of mismatch repair genes hints at possible paths by which tumorigenesis may proceed. To begin, upon inheritance of a mutant allele, the unimpaired copy of the gene is thought to supply normal mismatch function to the cell in most cases. A somatic mutation may later arise in this intact chromosomal copy, disabling the mismatch repair function of the cell. The elevated mutation rate that results could affect tumorigenesis in at least two ways. First, the overall increase in DNA damage could enhance the likelihood of inducing mutations in tumor suppressor genes and/or oncogenes, indirectly facilitating growth of tumors. In this regard, a noteworthy observation is that five of six mutational hotspots in the p53 gene are CpG sites where the cytosines are methylated to 5-methyIcytosine (72,113,114). As discussed earlier, MutS and MutL may be important for restoring Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 G-C base pairs when deamination of 5-methylcytosine changes this modified base into thymine, forming the G-T mispair. Thus, these repair genes may be critical in suppressing mutations at these hotspots. Second, it is possible that certain crucial genes are acutely prone to replication defects in the absence of mismatch repair. For example, Markowitz et al. (775) have shown that a significant correlation exists between colon cancer cell lines that exhibit microsatellite instability and the occurrence of mutations in a repetitive tract of the type II TGF-p receptor gene that contains 10 successive adenine residues (A10). This observation, combined with previous demonstrations that TGF-p mediates growth inhibition, led Markowitz et al. (775) to conclude that the TGF-P receptor mutations induced in these repair-deficient cells constitute "a critical step in tumor progression." The tissue specificity of TGF-p-mediated inhibition (776-720), moreover, illustrates how mismatch repair-related tumors could manifest in specific tissues. Considering the broad manifestations of HNPCC, it is important to point out that any of the numerous cellular roles of mismatch repair could be involved in preventing tumorigenesis. While the mutation avoidance function of mismatch repair genes implies clear routes by which tumors may develop in the absence of repair, less obvious possibilities remain to be explored. For example, the role of mismatch repair proteins in recombination and tumorigenesis, as well as the effects of these proteins on cell cycle checkpoints, is only beginning to be explored. It is conceivable that aberrant recombination events in mismatch repair-defective cells may lead to abnormal gene regulation and cancer. On the other hand, one can imagine that these events, by causing genomic instability, may eventually direct a cell toward premature lethality rather than immortality. This possibility is interesting, inasmuch as the prognosis for colorectal tumors with microsatellite instability may be more favorable than that of repair-competent tumors (727). Practical Conclusions The informed speculations discussed above reflect the enormous leap in understanding provided by illumination of the molecular basis of HNPCC. While these hypotheses may still require development and proof, they promise to produce insights into the pathways that lead to tumorigenesis. At the very least, the demonstration that some sporadic colorectal cancers have defects in mismatch repair reiterates the importance of this pathway. Some information gleaned from molecular studies can already be applied to clinical procedure: These studies have identified objective molecular criteria [to replace the controversial "Amsterdam criteria" (84)], which can now be used to classify cases of HNPCC. Application of these standards may help define physiologic defects associated with specific molecular lesions, distinguishing categories of HNPCC that have previously eluded detection and informing differential treatments. Realistically, routine application of these molecular standards in patient screening and prognosis may yet depend on technical advances that facilitate DNA sequencing. Moreover, conclusions based on screening should be predicated on extensive epidemiologic studies that address the penetrance of these mutaJournal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996 tions and the predictability of such screening procedures, both in affected families and in the general population (since penetrance and predictability may differ among the two). Unanswered Questions Over the long term, the insights into the molecular basis of HNPCC may answer some remaining questions about the disease: If all cells require mismatch repair, why are the tumors restricted to particular tissue types? Even within the colon, why do HNPCC tumors occur preferentially in the proximal colon? Why is HNPCC less aggressive than sporadic colorectal tumors? What, if any, is the role of the multiple homologues of MutLI Recently, mouse models have been developed for MSH2 deficiency and PMS2 deficiency (122,123). 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Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell 1995; 82:321-30. Notes Editor's note. Because of the large number of different proteins and genes discussed, all genes are italicized in this article. I would like to express my appreciation to Drs. Richard Kolodner and Bert Vogelstein for their helpful critiques of this manuscript. I am furthermore grateful to them and to Dr. Paul Modrich for providing unpublished materials. SPECIAL ARTICLE 251