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

Abstract

BackgroundThe 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.ResultsOur 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 BLM-dependent 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 WRN- and BLM-dependent unwinding was also re-examined in light of its possible role in preventing re-annealing. 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.ConclusionThese findings indicate that measurements of unwinding by WRN, BLM, and probably other RecQ helicases are complicated by their annealing properties. Thus, WRN- and BLM-dependent 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.

Mechanisms by Which Bloom Protein Can Disrupt Recombination Intermediates of Okazaki Fragment Maturation

“…Un-annealed regions are in boldface. ing of single-stranded DNA in the presence of annealing advocates Werner protein and BLM (34). Our results demonstrate that RPA promotes strand unwinding as well.…”

DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase

“…Besides helicase activity, several recent studies showed enhanced complementary DNA strand annealing by BLM and other RecQ helicases. Such activity has been demonstrated for BLM (9), WRN (10,11), RecQ5β (12), and RecQ1 (13). Strand annealing occurs most efficiently in the absence of ATP and is inhibited by single-strand binding proteins (SSBs) such as Replication Protein A (RPA) and SSB.…”

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

“…Where it has been mapped, this SA activity localizes to a region C-terminal of the core helicase domain near the less well-conserved RQC and HRDC domains (e.g., BLM residues 1290–1350) [36, 38]. This SA activity is inhibited by ATP and non-hydrolyzable nucleotide analogs [34–36, 38, 39] as well as by the ssDNA binding protein, Replication Protein A (RPA) [34, 36, 38, 40]. RPA is known to functionally interact with and stimulate the DNA helicase activity of BLM and WRN [40–43] where it binds through its 70 kDa subunit to the N-terminus of WRN and BLM [44].…”