Function of the Bloom's syndrome DNA helicase in the maintainance of genome integrity

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PROJECT SUMMARY The RecQ-like DNA helicase BLM is known for its critical role in the response to and repair of DNA-double- strand breaks in mammalian cells. Disruption of BLM activity causes Bloom’s syndrome, which is characterized by extreme cancer risk, short stature, and an average life expectancy of 25 years. Cancer susceptibility, chromosome breakage and other cellular defects are currently explained by the lack of BLM’s activity in the DNA-damage response and homologous recombination. In this proposal we are testing the hypothesis that BLM plays critical roles in DNA replication initiation and elongation to maintain chromosome stability in unperturbed cells. This hypothesis is based on extensive preliminary data, including an unbiased screen of the mid-S-phase proteome that led to the discovery that chromatin-bound BLM directly interacts with the Mcm6 subunit of chromatin-bound Mcm2-7. Notably, two distinct binding sites in BLM and Mcm6 differentially regulate complex formation in G1 and S-phase, and disruption of the BLM/Mcm6 interaction in S-phase, but not in G1, leads to supra-normal DNA replication speed. Aberrant acceleration of DNA replication speed beyond a safe limit is emerging as a mechanism that causes DNA damage and kills certain types of cancer cells. Our preliminary findings suggest that the BLM/Mcm6 interaction acts as a novel, negative regulator of DNA replication in human cells. That cells lacking BLM do not exhibit increased replication speed suggests that acceleration of replication requires the BLM protein, leading us to hypothesize that BLM needs to be tethered to Mcm6 to restrict the ATPase/helicase activity of BLM to the immediate vicinity of the replisome. Together with BLM’s ability to unwind G-quadruplexes (G4s) and their presence throughout the human genome, including at ~90% of origins of replication, we propose that BLM is recruited by Mcm6 to unfold DNA structures (i) at replication origins to facilitate the G1/S transition (Aim 1) and (ii) throughout the genome to regulate replisome progression during unperturbed S-phase (Aim 2). We have isolated a set of BLM mutants that specifically fail to interact with Mcm6 in G1 or S-phase, or both, to identify the separate functions of the BLM/Mcm6 interaction in G1 and S-phase and to determine replication-associated mitotic defects. Further, we will use biophysical approaches and molecular dynamics simulations to determine the mechanism of G4 unwinding by BLM (Aim 3). Completing these studies will delineate a major new function for BLM in unperturbed DNA replication, besides its established role in DNA double-strand break repair and replication fork restart after DNA damage, and determine its mechanism of G4 unwinding. Our findings will provide a major advance in our understanding of the mechanisms that prevent chromosome instability in unperturbed cells and improve our understanding of chromosome breakage syndromes and cancer predisposition.