Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

Roles of mismatch repair proteins in the recognition and processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) are among the most toxic type of damage to a cell. Many ICL-inducing agents are widely used as therapeutic agents, e.g. cisplatin, psoralen. A bettor understanding of the cellular mechanism that eliminates ICLs is important for the improvement of human health. However, ICL repair is still poorly understood in mammals. Using a triplex-directed site-specific ICL model, we studied the roles of mismatch repair (MMR) proteins in ICL repair in human cells. We are also interested in using psoralen-conjugated triplex-forming oligonucleotides (TFOs) to direct ICLs to a specific site in targeted DNA and in the mammalian genomes. ^ MSH2 protein is the common subunit of two MMR recognition complexes, and MutSα and MutSβ. We showed that MSH2 deficiency renders human cell hypersensitive to psoralen ICLs. MMR recognition complexes bind specifically to triplex-directed psoralen ICLs in vitro. Together with the fact that psoralen ICL-induced repair synthesis is dramatically decreased in MSH2 deficient cell extracts, we demonstrated that MSH2 function is critical for the recognition and processing of psoralen ICLs in human cells. Interestingly, lack of MSH2 does not reduce the level of psoralen ICL-induced mutagenesis in human cells, suggesting that MSH2 does not contribute to error-generating repair of psoralen ICLs, and therefore, may represent a novel error-free mechanism for repairing ICLs. We also studied the role of MLH1, anther key protein in MMR, in the processing of psoralen ICLs. MLH1-deficient human cells are more resistant to psoralen plus UVA treatment. Importantly, MLH1 function is not required for the mutagenic repair of psoralen ICLs, suggesting that it is not involved in the error-generating repair of this type of DNA damage in human cells. ^ These are the first data indicating mismatch repair proteins may participate in a relatively error-free mechanism for processing psoralen ICL in human cells. Enhancement of MMR protein function relative to nucleotide excision repair proteins may reduce the mutagenesis caused by DNA ICLs in humans. ^ In order to specifically target ICLs to mammalian genes, we identified novel TFO target sequences in mouse and human genomes. Using this information, many critical mammalian genes can now be targeted by TFOs.^

Mismatch Repair Deficient Tumors Lacking Known Sporadic Causes: Are They All Due to Lynch Syndrome?

BACKGROUND: Mismatch repair deficient (MMRD) colorectal (CRC) or endometrial (EC) cancers in the absence of MLH1 promoter hypermethylation and BRAF mutations are suggestive of Lynch syndrome (LS). Positive germline genetic test results confirm LS. It is unclear if individuals with MMRD tumors but no identified germline mutation or sporadic cause (MMRD+/germline-) have LS. HYPOTHESIS: Since LS is hereditary, individuals with LS should have a stronger family history of LS-related cancers than individuals with sporadic tumors. We hypothesized that MMRD+/germline- CRC and/or EC patients would have less suggestive family histories than LS CRC and/or EC patients. METHODS: 253 individuals with an MMRD CRC or EC who underwent genetic counseling at one institution were included in analysis in 1 of 4 groups: LS, MMRD+/germline-, MMRD+/VUS, sporadic MSI-H (MMRD tumor with MLH1 promoter hypermethylation or BRAF mutation). Family histories were analyzed utilizing MMRpro and PREMM1,2,6. Kruskal-Wallis tests were used to compare family history scores. Logistic regression was used to determine what factors were predictive of LS. RESULTS: MMRD+/germline- individuals had significantly lower median family history scores (PREMM1,2,6=7.3, MMRpro=8.1) than LS individuals (PREMM1,2,6=26.1, MMRpro=89.8, p CONCLUSION: MMRD+/germline- individuals have less suggestive family histories of LS than individuals with LS, but more suggestive family histories than sporadic MSI-H individuals. CRC and/or EC patients with abnormal tumor studies are more likely to have a germline LS mutation if they have a family history suggestive of hereditary cancer. These results imply that the MMRD+/germline- group may not all have LS. This finding highlights the need to determine other somatic, epigenetic or germline causes of MMRD tumors so that these patients and their families can be accurately counseled regarding screening and management.