Measurement of DNA mismatch repair activity in live cells

Loss of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Currently, assays for DNA MMR activity involve the use of cell extracts and are technically challenging and costly. Here, we report a rapid, less labor-intensive method that can quantitatively measure MMR activity in live cells. A G–G or T–G mismatch was introduced into the ATG start codon of the enhanced green fluorescent protein (EGFP) gene. Repair of the G–G or T–G mismatch to G–C or T–A, respectively, in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were transfected in parallel into the same cell line and the number of green cells counted by flow cytometry. Relative EGFP expression was calculated as the total fluorescence intensity of cells transfected with the heteroduplex construct divided by that of cells transfected with the homoduplex construct. We have tested several cell lines from both MMR-deficient and MMR-proficient groups using this method, including a colon carcinoma cell line HCT116 with defective hMLH1 gene and a derivative complemented by transient transfection with hMLH1 cDNA. Results show that MMR-proficient cells have significantly higher EGFP expression than MMR-deficient cells, and that transient expression of hMLH1 alone can elevate MMR activity in HCT116 cells. This method is potentially useful in comparing and monitoring MMR activity in live cells under various growth conditions.     

Acetylation regulates DNA repair mechanisms in human cells

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.     

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA. Finally, pharmacologic or genetic inhibition of p38alpha increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G(2) checkpoint bypass. These results suggest that p38alpha links DNA MMR to the G(2) checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.     

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.     

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3-6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.     

Evidence That Nucleosomes Inhibit Mismatch Repair in Eukaryotic Cells

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSα and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSα specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSα displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSα sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.     

Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal

The Saccharomyces cerevisiae mismatch repair (MMR) protein MSH6 and the SGS1 helicase were recently shown to play similarly important roles in preventing recombination between divergent DNA sequences in a single-strand annealing (SSA) assay. In contrast, MMR factors such as Mlh1p, Pms1p, and Exo1p were shown to not be required or to play only minimal roles. In this study we tested mutations that disrupt Sgs1p helicase activity, Msh2p-Msh6p mismatch recognition, and ATP binding and hydrolysis activities for their effect on preventing recombination between divergent DNA sequences (heteroduplex rejection) during SSA. The results support a model in which the Msh proteins act with Sgs1p to unwind DNA recombination intermediates containing mismatches. Importantly, msh2 mutants that displayed separation-of-function phenotypes with respect to nonhomologous tail removal during SSA and heteroduplex rejection were characterized. These studies suggest that nonhomologous tail removal is a separate function of Msh proteins that is likely to involve a distinct DNA binding activity. The involvement of Sgs1p in heteroduplex rejection but not nonhomologous tail removal further illustrates that subsets of MMR proteins collaborate with factors in different DNA repair pathways to maintain genome stability.     

Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.     

Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within activin receptor 2 (ACVR2)

We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.     

Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.     

Steady-state ATPase activity of E. coli MutS modulated by its dissociation from heteroduplex DNA

The ability of MutS to recognize mismatched DNA is required to initiate a mismatch repair (MMR) system. ATP binding and hydrolysis are essential in this process, but their role in MMR is still not fully understood. In this study, steady-state ATPase activities of MutS from Escherichia coli were investigated using the spectrophotometric method with a double end-blocked heteroduplex containing gapped bases. The ATPase activities of MutS increased as the number of gapped bases increased in a double end-blocked heteroduplex with 2-8 gapped bases in the chain, indicating that MutS dissociates from DNA when it reaches a scission during movement along the DNA. Since movement of MutS along the chain does not require extensive ATP hydrolysis and the ATPase activity is only enhanced when MutS dissociates from a heteroduplex, these results support the sliding clamp model in which ATP binding by MutS induces the formation of a hydrolysis-independent sliding clamp.     

Selective cytotoxicity of rhodium metalloinsertors in mismatch repair-deficient cells

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.     

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.     

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.     

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.     

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.