Thesis
Functional characterization of human hMRE11 and hMRE11-hMLH1 interplay in DNA mismatch repair
Washington State University
Master of Science (MS), Washington State University
2006
Handle:
https://hdl.handle.net/2376/446
Abstract
DNA mismatch repair (MMR) is a critical pathway which can maintain genetic stability by correcting mismatched nucleotides that occur during DNA replication and recombination. Cells that are deficient in MMR display microsatellite instability (MSI) and germline mutations in various MMR genes can lead to hereditary nonpolyposis colorectal cancer (HNPCC) in humans. Our study for the first time showed that hMRE11 deficiency in HeLa cells can lead to defective 3' MMR in an in vitro MMR assay. Through both GFP-based MSI reporter assay and endogenous MSI marker assay, these cells were also found to display increased levels of MSI, which is comparable to that of the hMLH1 deficient cells. Together with our previous finding that hMRE11 directly interacts with hMLH1, we raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1, and this interaction is critical for functional MMR. Our analysis with 7 hMLH1 HNPCC mutations located within the hMRE11-interacting domain showed that 4 of them almost completely disrupt the MRE11-MLH1 interaction. We increased our study to 64 MLH1 HNPCC mutations, which contain 38 missense mutations, and the result showed that 63% (24 out of 38) of the hMLH1 missense mutations can disrupt their interactions with hMRE11. These findings indicate that hMRE11 represents a functional protein factor in MMR pathway and disruption of hMRE11-hMLH1 could be an alternative molecular mechanism underlying the pathogenic effects of hMLH1 HNPCC mutations. To directly study the functional impact of hMRE11-hMLH1 interplay in MMR, we blocked their interaction in vivo by an hMRE11 dominant negative mutant which represents the hMLH1-interacting domain. In vitro MMR and excision assays indicate that these cells showed dramatically decreased 3' MMR activity and 3' excision activity, suggesting that the hMRE11-hMLH1 interplay is critical for functional MMR, presumably by recruiting hMRE11 for the excision step of MMR.
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Details
- Title
- Functional characterization of human hMRE11 and hMRE11-hMLH1 interplay in DNA mismatch repair
- Creators
- Fengxue Zhu
- Contributors
- Chengtao Her (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525011801842
- Language
- English
- Resource Type
- Thesis