Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutL␣ (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X) 2 E(X) 4 E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutL␣, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutL␣ is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutS␣, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X) 2 E(X) 4 E motif abolishes yMutL␣ endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutL␣ ATPase activity or the ability of the protein to support assembly of the yMutL␣⅐yMutS␣⅐heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutL␣ incision to the discontinuous strand of a nicked heteroduplex.

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.MutLalpha | genetic instability | cancer | triplet repeats

MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.

Mismatch repair contributes to genetic stability, and inactivation of the mammalian pathway leads to tumor development. Mismatch correction occurs by an excision-repair mechanism and has been shown to depend on the 5 to 3 hydrolytic activity exonuclease 1 (Exo1) in eukaryotic cells. However, genetic and biochemical studies have indicated that one or more Exo1-independent modes of mismatch repair also exist. We have analyzed repair of nicked circular heteroduplex DNA in extracts of Exo1-deficient mouse embryo fibroblast cells. Exo1-independent repair under these conditions is MutL␣-dependent and requires functional integrity of the MutL␣ endonuclease metal-binding motif. In contrast to the Exo1-dependent reaction, we have been unable to detect a gapped excision intermediate in Exo1-deficient extracts when repair DNA synthesis is blocked. A possible explanation for this finding has been provided by analysis of a purified system comprised of MutS␣, MutL␣, replication factor C, proliferating cell nuclear antigen, replication protein A, and DNA polymerase ␦ that supports Exo1-independent repair in vitro. Repair in this system depends on MutL␣ incision of the nicked heteroduplex strand and dNTPdependent synthesis-driven displacement of a DNA segment spanning the mismatch. Such a mechanism may account, at least in part, for the Exo1-independent repair that occurs in eukaryotic cells, and hence the modest cancer predisposition of Exo1-deficient mammalian cells.cancer ͉ DNA polymerase ͉ DNA repair ͉ strand displacment

Bidirectional mismatch repair directed by a strand break located 3 or 5 to the mispair has been reconstituted using seven purified human activities: MutS␣, MutL␣, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase ␦. In addition to DNA polymerase ␦, PCNA, RFC, and RPA, 5-directed repair depends on MutS␣ and EXOI, whereas 3-directed mismatch correction also requires MutL␣. The repair reaction displays specificity for DNA polymerase ␦, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase ␦ in the excision step of repair. RFC and PCNA dramatically activate polymerase ␦-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase ␦ are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3-and 5-directed excision in HeLa nuclear extracts by only 20 -30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.Mismatch repair stabilizes the genome by correction of DNA biosynthetic errors, by ensuring the fidelity of genetic recombination, and in mammalian cells by participation in the cellular response to some classes of DNA damage. Basic features of the reaction responsible for replication error correction are conserved from bacteria to mammals. Study of partial reactions has demonstrated that repair can be divided into three major steps: mismatch recognition, excision, and repair DNA synthesis (1-4). Escherichia coli mismatch repair has been reconstituted using purified components. In the bacterial reaction MutS is responsible for mismatch recognition and recruits MutL to the heteroduplex in an ATP-dependent manner (5-13). Assembly of the MutL⅐MutS⅐heteroduplex complex activates the d(GATC) endonuclease activity of MutH, which incises the unmethylated strand of the heteroduplex (14). This strand break serves as an entry point for the excision system, which is comprised of DNA helicase II and an appropriate single strand exonuclease (15)(16)(17)(18). A 3Ј to 5Ј exonuclease is required when the nick that directs excision is located 3Ј to the mismatch, whereas a 5Ј to 3Ј hydrolytic activity is necessary when the strand break is 5Ј to the mispair. DNA polymerase III holoenzyme is sufficient to repair the ensuing gap, and covalent integrity is restored to the helix by DNA ligase (19).Analysis of nuclear extracts of human cells has indicated a similar exci...

DNA mismatch repair (MMR) is a multifunctional process that promotes genetic stability and suppresses carcinogenesis. Correction of DNA replication errors is its major function. Despite the importance of MMR, its functioning in eukaryotes is not well understood. Here we report that human mismatch correction reactions in cell-free extracts occur during concomitant nick-dependent nucleosome assembly shaped by the replication histone chaperone CAF-I. Concomitant nucleosome assembly protects the discontinuous mismatch-containing strands from excessive degradation by MMR machinery. Such protection is also demonstrated in a defined purified system that supports both mismatch correction and CAF-I-dependent histone H3–H4 deposition reactions. In addition, we find that the mismatch recognition factor MutSα suppresses CAF-I-dependent histone H3–H4 deposition in a mismatch-dependent manner. We suggest that there is active crosstalk between MMR and replication-dependent nucleosome assembly during the correction of DNA replication errors and, as a result, the nascent mismatch-containing strands are degraded in a controlled manner.

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1. MMR has evolved to process mismatches arising during replication or recombination (1, 2). That its malfunction leads to cancer (3) bears witness to the importance of its role in the maintenance of genomic stability. In eukaryotes, mismatch repair (MMR) is initiated by the binding of MutSα, a heterodimer of MSH2 and MSH6, to non-Watson-Crick base pairs in DNA. Exchange of ADP for ATP then converts MutSα into a sliding clamp, which diffuses along the DNA contour, possibly together with a heterodimer of MLH1/PMS2 named MutLα, in search of free DNA termini that serve as initiation sites for exonuclease 1-catalyzed degradation of the error-containing DNA strand (1, 2).In mammalian cells, free termini are generally marked by PCNA, which helps orchestrate replication and repair DNA synthesis through interactions with key players of DNA metabolism, including MSH6 and chromatin assembly factor 1 (CAF-1) (4). The latter complex, composed of p150, p60, and p48 subunits (5), promotes rapid assembly of nucleosomes on newly replicated DNA (6).Nucleosome loading causes supercoiling, which compacts the genome and increases its stability, but which makes DNA less accessible to metabolic processes. Thus, during nucleotide excision repair, efficient processing of UV-induced DNA damage requires chromatin remodeling (7, 8) and CAF-1-mediated chromatin reassembly upon repair completion (9). Whether similar transactions take place during MMR is unknown. As the sliding function of MutSα was reported to be blocked by nucleosomes (10, 11), albeit not in all sequence contexts (12), we argued that mismatches arising during replication would have to be repaired ...