Biochemical basis of WRN and RecQ helicase function

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DESCRIPTION (provided by applicant): RecQ helicases act to maintain genomic stability by an as yet unknown mechanism. When their function is lost, levels of illegitimate recombination increase significantly. Not surprisingly, the human hereditary RecQ deficiency diseases (Werner, Bloom, and Rothmund-Thomson syndromes, caused by defects in WRN, BLM, and RECQL4, respectively) demonstrate early onset and increased frequency of cancer. Importantly, Werner syndrome also shows accelerated development of many age-related problems. Although these diseases have distinct phenotypes, RecQ family members maintain a high degree of homology within and C terminal to the conserved central helicase domain, suggesting that they may have a common mechanistic function and/or DNA substrate specificity. We refer to this extended sequence conservation as the RecQ expanded core. Their illegitimate recombination phenotypes suggest that RecQ helicases function in recombination or anti-recombination pathways, or possibly in resolution of replication fork blockage. Our laboratory has recently uncovered strand pairing and strand exchange activities in WRN and other RecQ helicases consistent with putative roles in these pathways. We hypothesize that the RecQ expanded core forms a functional unit that encompasses DNA binding and catalytic activities. Further, we propose that the function of the RecQ expanded core is to coordinate DNA binding and unwinding to achieve strand exchange reactions in complex recombination or replication intermediates. This hypothesis fits with the putative roles for RecQ members and current biochemical knowledge regarding these proteins. In this proposal, WRN is used as a model RecQ helicase for 1) characterizing strand exchange activity reflecting putative coordination between strand pairing and unwinding activities, 2) examining DNA binding properties and substrate specificity for replication and recombination intermediates, and 3) generating site-directed mutants that pinpoint the DNA binding, enzymatic, and physiological functions of the RecQ expanded core and its individual domains. Our findings with WRN will be highly relevant to elucidating its DNA metabolic role and specific mechanisms underlying carcinogenesis and certain aging phenotypes. Sequence conservation between RecQ helicases also indicates that our findings will be applicable to the functions of other RecQ helicases (including BLM and RECQL4) and their relationships to human health.