This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The RecQ helicase gene, BLM, encodes one of five RecQ helicases in human cells. Mutations in the genes encoding three of these helicases, WRN, RECQ4, and BLM, cause diseases characterized by premature aging and/or cancer predisposition. Homozygous mutations in BLM lead to the cancer predisposition disorder known as Bloom syndrome. Thus, BLM is classified as a caretaker tumor suppressor. Bloom patients are prone to almost all classes of cancer. Knowledge of the specific mechanisms by which BLM protects the genome, despite years of general characterization, is incomplete due to the complexity of the underlying processes at the replication fork and our limited understanding of the replication machinery in eukaryotes. In this machinery, additional DNA helicases perform essential functions. We have found that the human BLM tumor suppressor gene functions in yeast to suppress both the DNA replication and DNA repair defects in mutants defective in another helicase, Dna2 helicase. We propose to use this observation to understand human BLM and how it preserves genome integrity. Dna2 is involved in Okazaki fragment processing, a critical step for maintaining high fidelity DNA replication. Suppression of dna2 mutants therefore suggests for the first time that BLM plays a critical role in Okazaki fragment processing (OFP). We believe that due to the complexity of replisomes and repairosomes, the only sure way to address the relevance of the suppression of dna2 by human BLM is to use a defined replication/repair system consisting entirely of purified proteins and defined oligonucleotide substrates that mimic intermediates in OFP. We will use biochemical reconstitution of the various stages of Okazaki fragment processing to identify the specific role of the BLM helicase. To take advantage of the simpler milieu in which replication and recombination take place in the unicellular eukaryote, yeast, we have limited our studies to date to the yeast model system, but will now begin to extend our studies to human cells. We will investigate the interaction between the replicative Dna2 helicase and Bloom in human cells directly. We will carry out cell biological analysis of the localization of human Dna2, focusing on telomeres, since in yeast Dna2 is dynamically localized to telomeres.
Showing the most recent 10 out of 583 publications
