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Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1

Langland, Gregory Todd
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052316756

Abstract Details

2003, PhD, University of Cincinnati, Medicine : Molecular Genetics, Biochemistry and Microbiology.
Bloom's syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes affected individuals to cancer. Such individuals also are small in size, sensitive to the sun, have immune dysfunction and gross genomic instability. The cytogenetics of BS cells have been extensively studied and have shown increased levels of homologous recombination, quadriradial formations, telomeric associations and chromosome breakage. The gene responsible for BS has been positionally cloned and and encodes a RecQ helicase family member with strand displacement activity that is dependent on ATP and Mg2+. In order to have a greater understanding of BLM helicase function in the cell in regards to DNA replication, recombination and repair, we identified protein-partners of BLM. The C-terminus of BLM identified the DNA mismatch repair protein MLH1 from a yeast two-hybrid screen. In vitro and in vivo immunoprecipitations confirmed the interaction between these two proteins. Using an in vitro mismatch repair assay, BS cell extracts were tested for their ability to correct a single nucleotide mismatch. The BS cell extracts were able to remove the single nucleotide mismatch from the plasmid DNA, demonstrating that the BLM-MLH1 interaction is not necessary to correct a single nucleotide mismatch substrates. Helicase assays then were performed which demonstrated that MLH1 or the mutL heterodimer modulates the enzymatic activity of BLM by stimulating BLM's strand displacement activity on the double-overhang (DO) substrate. Finally, we performed experiments with the supF20 mutagenesis system and demonstrated that extracts from BS cells are unable to utilize micro-homology elements within the supF20 gene to restore supF function following the induction of a double strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable to those of normal extracts when ligatable ends are present, a significant 5-fold increase in mutation rate with BS extracts is observed when terminal phosphates are removed from the DNA substrate that needs repair. Mutant plasmids recovered following DSB repair by BS extracts contain smaller deletions within the lacZ α gene not commonly recovered from normal extracts. Colorectal cancer cell lineHCT116 extracts lacking MLH1 were also examined although the efficiency and fidelity of end-joining was similar to control extracts. This suggests that the BLM-MLH1 interaction is not necessary for proper end-joining. In summary this work demonstrates that BS cells lacking the BLM helicase process DSBs differently than normal cells and strongly suggests a role for BLM in aligning micro-homology elements during recombinational events in DSB repair. Disruption of the BLM helicase may lead to replication fork collapses, improper processing of DSBs, genomic instability and ultimately cancer.
Dr. Joanna Groden (Advisor)
133 p.
Chemistry, Biochemistry
Bloom's Syndrome; RecQ Helicase; DNA repair; Homologous Recombination; mismatch repair

Recommended Citations

Citations

  • Langland, G. T. (2003). Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1 [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052316756

    APA Style (7th edition)

  • Langland, Gregory. Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1. 2003. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052316756.

    MLA Style (8th edition)

  • Langland, Gregory. "Interaction Between the BLM Helicase and the DNA Mismatch Repair Protein, MLH1." Doctoral dissertation, University of Cincinnati, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1052316756

    Chicago Manual of Style (17th edition)

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© 2003, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.