Andrew B. Buermeyer

Defects in MLH1, as with other mismatch repair (MMR) proteins, are the primary cause of hereditary nonpolyposis colon cancer (HNPCC). Mutations in MMR genes often disrupt mismatch repair and MMR signaling functions. However, some HNPCC-associated mutations have unknown pathogenicity. Here, we uncover an MLH1 clinical mutation with a leucine (L)-to-histidine (H) amino acid change at position 607 that ablates MLH1 binding to FANCJ. Given that a DNA helicase is not essential for mammalian MMR in vitro, we considered that loss of MLH1 binding to FANCJ could alter MMR signaling. Consistent with this hypothesis, FANCJ-deficient cells exhibit delayed MMR signaling and apoptotic responses that generate resistance to agents that induce O⁶-methylguanine lesions. Our data indicate that the delay in MMR signaling provides time for the methylguanine methyltransferase (MGMT) enzyme to reverse DNA methylation. In essence, FANCJ deficiency alters the competition between two pathways: MGMT-prosurvival versus MMR-prodeath. This outcome could explain the HNPCC familial cancers that present as microsatellite stable and with intact MMR, such as MLH^L607H. Importantly, the link between FANCJ and HNPCC provides insight toward directed therapies because loss of the FANCJ/MLH1 interaction also uniquely sensitizes cells to DNA cross-linking agents. Cancer Prev Res; 3(11); 1409–16. ©2010 AACR.

Background: Methods: Results: Conclusion:

▪ Abstract DNA mismatch repair (MMR) is one of multiple replication, repair, and recombination processes that are required to maintain genomic stability in prokaryotes and eukaryotes. In the wake of the discoveries that hereditary nonpolyposis colorectal cancer (HNPCC) and other human cancers are associated with mutations in MMR genes, intensive efforts are under way to elucidate the biochemical functions of mammalian MutS and MutL homologs, and the consequences of defects in these genes. Genetic studies in cultured mammalian cells and mice are proving to be instrumental in defining the relationship between the functions of MMR in mutation and tumor avoidance. Furthermore, these approaches have raised awareness that MMR homologs contribute to DNA damage surveillance, transcription-coupled repair, and recombinogenic and meiotic processes.

Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the Pms2 or Mlh1 MMR gene develop cancer but exhibit very different tumor spectra; only Mlh1 ^−/− animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an in vivo forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in Mlh1 ^−/− mice was 2- to 3-fold higher than in Pms2 ^−/− animals. The higher mutation frequency in Mlh1 ^−/− mice may be a consequence of some residual DNA repair capacity in the Pms2 ^−/− animals. Relevant to this idea, we observed that Pms2 ^−/− mice exhibit almost normal levels of Mlh1p, whereas Mlh1 ^−/− animals lack both Mlh1p and Pms2p. Comparison between Mlh1 ^−/− animals and Mlh1 ^−/− and Pms2 ^−/− double knockout mice revealed little difference in mutator phenotype, suggesting that Mlh1 nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of Mlh1 ^−/− mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.