Helicase regulation during homologous recombination

Columbia University Health Sciences, New York NY

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Abstract

Project Summary Our chromosomes are continually bombarded with a variety of insults, resulting in damage that must be repaired. By necessity, cells have evolved mechanisms to detect and repair broken strands of DNA, thereby preventing loss of important genetic information. Double-stranded DNA breaks (DSBs) are a type of damage that led to particularly disastrous outcomes. If not corrected, DSBs can lead to gross chromosomal rearrangements, which are the hallmark of all forms of cancer. Indeed, defects in HR-related proteins are associated with several severe genetic diseases. Patients with these diseases often exhibit a strong predisposition for developing cancers due to a loss of genome integrity. Surprisingly, DNA replication is the primary source of DSBs, and as a consequence rapidly growing cells are especially dependent upon homologous DNA recombination for survival. This dependence upon homologous recombination for the survival of rapidly growing cells highlights the potential for using recombination inhibitors as highly selective cancer therapies. To fully exploit the clinical potential of homologous recombination inhibitors it will be essential that we more fully understand the detail molecular underpinnings of recombination and the proteins that are involved in regulating and controlling this process. To help better understand the molecular basis of homologous DNA recombination we have developed powerful new experimental platforms that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level in real time. We are utilizing these unique research tools to probe the fundamental basis for protein-nucleic acid interactions, with emphasis placed upon understanding reactions relevant to human biology and disease. Here we will assess how members of the RECQ family of ATP-dependent helicases function during long range resection of double stranded DNA breaks. Our work will focus on the human RECQ helicases BLM and WRN, both of which are associated with cancer and cancer predisposition syndromes. We will define how the end resection machineries are assembled onto DNA ends, we will map protein-protein and protein-DNA interactions that are required for end processing, we will determine how end resection is coupled to downstream steps in homologous recombination, and we will test disease relevant mutations to fully define their molecular defects. We will seek to determine detailed molecular information related to these questions, and part of the significance of this project lies in the depth of the answers we strive to obtain.