Competition between the DNA unwinding and strand pairing activities of the Werner and Bloom syndrome proteins

摘要

Background: The premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM,respectively.Recently,both WRN and BLM (as well as several other RecQ members) have been shown to possess a strand annealing activity in addition to the requisite DNA unwinding activity.Since an annealing function would appear to directly oppose the action of a helicase,we have examined in this study the dynamic equilibrium between unwinding and annealing mediated by either WRN or BLM.Results: Our investigation into the competition between annealing and unwinding demonstrates that,under standard reaction conditions,WRN- or BLM-mediated annealing can partially or completely mask unwinding as measured in standard helicase assays.Several strategies were employed to suppress the annealing activity so that the actual strength of WRN- or BLMdependent unwinding could be more accurately assessed.Interestingly,if a DNA oligomer complementary to one strand of the DNA substrate to be unwound is added during the helicase reaction,both WRN and BLM unwinding is enhanced,presumably by preventing protein-mediated re-annealing.This strategy allowed measurement of WRN-catalyzed unwinding of long (80 base pair) duplex regions and fully complementary,blunt-ended duplexes,both of which were otherwise quite refractory to the helicase activity of WRN.Similarly,the addition of trap strand stimulated the ability of BLM to unwind long and blunt-ended duplexes.The stimulatory effect of the human replication protein A (hRPA,the eukaryotic single-stranded DNA binding protein) on both WRNand BLM-dependent unwinding was also re-examined in light of its possible role in preventing reannealing.Our results show that hRPA influences the outcome of WRN and BLM helicase assays by both inhibiting re-annealing and directly promoting unwinding,with the larger contribution from the latter mechanism.Conclusion: These findings indicate that measurements of unwinding by WRN,BLM,and probably other RecQ helicases are complicated by their annealing properties.Thus,WRN- and BLMdependent unwinding activities are significantly stronger than previously believed.Since this broadens the range of potential physiological substrates for WRN and BLM,our findings have relevance for understanding their functions in vitro and in vivo.