Paul Modrich

Hypermutability and mismatch repair deficiency in RER+ tumor cells

A subset of sporadic colorectal tumors and most tumors developing in hereditary nonpolyposis colorectal cancer patients display frequent alterations in microsatellite sequences. Such tumors have been thought to manifest replication errors (RER+), but the basis for the alterations has remained conjectural. We demonstrate that the mutation rate of (CA)n repeats in RER+ tumor cells is at least 100-fold that in RER- tumor cells and show by in vitro assay that increased mutability of RER+ cells is associated with a profound defect in strand-specific mismatch repair. This deficiency was observed with microsatellite heteroduplexes as well as with heteroduplexes containing single base-base mismatches and affected an early step in the repair pathway. Thus, a true mutator phenotype exists in a subset of tumor cells, the responsible defect is likely to cause transitions and transversions in addition to microsatellite alterations, and a biochemical basis for this phenotype has been identified.

Endonucleolytic Function of MutLα in Human Mismatch Repair

Summary Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLα (MLH1•PMS2 heterodimer). MutLα is required for mismatch repair, but its function in this process is unclear. We show that human MutLα is a latent endonuclease that is activated in a mismatch-, MutSα-, 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 MutSα-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.

DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma

PURPOSE We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.

Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha.

A human MSH2-human MSH3 (hMSH2·hMSH3) complex of approximately 1:1 stoichiometry (human MutSβ (hMutSβ)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSβ supports the efficient repair of insertion/deletion (I/D) heterologies of 2–8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSα (hMutSα), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSβ. Thus, the repair specificities of hMutSα and hMutSβ are redundant with respect to the repair of I/D heterologies of 2–8 nucleotides. The hMutSα level in repair-proficient HeLa cells (1.5 μg/mg nuclear extract) is approximately 10 times that of hMutSβ. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSα, the hMutSβ level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSα and hMutSβ, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10144–10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.

Mechanisms in eukaryotic mismatch repair.

Inactivation of the human mismatch repair system confers a large increase in spontaneous mutability and a strong predisposition to tumor development. Mismatch repair provides several genetic stabilization functions: it corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination, and participates in the earliest steps of checkpoint and apoptotic responses to several classes of DNA damage (see refs. 1-3 for recent reviews). Defects in this pathway are the cause of typical and atypical hereditary nonpolyposis colon cancer (4), but may also play a role in the development of 15 to 25% of sporadic tumors that occur in a number of tissues (5). The system is also of biomedical interest because mismatch repair-deficient tumor cells are resistant to certain cytotoxic chemotherapeutic drugs (2,3), a manifestation of its involvement in the DNA damage response. Of the several mutation avoidance functions of mismatch repair, the reaction responsible for replication error correction has been the most thoroughly studied, and the discussion that follows is restricted to this pathway.

MutS mediates heteroduplex loop formation by a translocation mechanism.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein‐stabilized, α‐shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10 000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP‐dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl‐directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS‐mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of α‐loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Cisplatin and adriamycin resistance are associated with MutLα and mismatch repair deficiency in an ovarian tumor cell line

In contrast to parental A2780 ovarian tumor cells, extracts of one doxorubicin-resistant and two independent cis-diamminedichloroplatinum(II)-resistant derivatives are defective in strand-specific mismatch repair. The repair defect of the three hypermutable, drug-resistant cell lines is only evident when the strand break that directs the reaction is located 3′ to the mismatch, and in each case repair is restored to extracts by addition of purified MutLα heterodimer. As judged by immunological assay, drug resistance is associated with the virtual absence of the MutLα MLH1 subunit and greatly reduced levels of the PMS2 subunit. These findings implicate a functional mismatch repair system in the cytotoxic effects of these antitumor drugs and may have ramifications for their clinical application.

Mismatch repair deficiency in phenotypically normal human cells

Tumor cells in patients with hereditary nonpolyposis colorectal cancer (HNPCC) are characterized by a genetic hypermutability caused by defects in DNA mismatch repair. A subset of HNPCC patients was found to have widespread mutations not only in their tumors, but also in their non-neoplastic cells. Although these patients had numerous mutations in all tissues examined, they had very few tumors. The hypermutability was associated with a profound defect in mismatch repair at the biochemical level. These results have implications for the relation between mutagenesis and carcinogenesis, and they suggest that mismatch repair deficiency is compatible with normal human development.

Strand-specific Mismatch Repair in Mammalian Cells*

Mistakes occurring during DNA transactions necessarily produce mismatched base pairs. DNA biosynthetic errors that escape the polymerase editing function yield mispaired bases, but mismatches also populate a recombination heteroduplex produced by strand transfer between related sequences that have diverged at the nucleotide level. Study of microbial systems has demonstrated the importance of mismatch correction in the reversal of such errors and consequent stabilization of the genome (1‐ 4). 1 Interest in this process has increased dramatically over the past few years with the demonstration that mismatch repair plays an equally important role in mutation avoidance in mammalian cells and the finding that inactivation of the system by inherited or somatic mutation predisposes cells to tumor development and renders them resistant to the cytotoxic effects of certain DNA-damaging agents. This minireview will emphasize the molecular features of the mammalian strand-specific mismatch repair pathway. Work in other systems will be alluded to only as necessary for clarity or in those instances where compelling supporting data is available.

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5′→3′ double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.