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.
Highlights
The premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM, respectively
Our laboratory has demonstrated annealing activity associated with the human WRN and BLM proteins that are deficient in the cancer-prone diseases Werner syndrome (WS) and Bloom syndrome (BS), respectively [14]
When helicase assays were performed using one fourth the amount of substrate, significantly higher levels of unwinding of the 3' tailed 80 bp duplex can be detected without addition of the trap strand (Fig. 2F). These results are the first demonstration that WRN alone can unwind a duplex of at least 80 bp, and conclusively demonstrate that WRNand BLM-mediated re-annealing tends to directly counteract their abilities to unwind duplex substrates. These findings indicate that, to maximize unwinding, WRN or BLM helicase assays should be performed at the lowest possible DNA substrate concentration and suggest that previous experiments to determine BLM- or WRN-dependent unwinding of long duplexes may have been performed under conditions in which strand pairing mediated by these same proteins partially or completely masked unwinding
Summary
The premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM, respectively. 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. Helicases of the RecQ family play important roles in maintaining genomic stability, as loss of their individual functions results in chromosomal abnormalities due to increased illegitimate recombination [reviewed in [1,2]]. There are five RecQ helicases in humans, including the WRN, BLM, and RecQ4 proteins that are defective in Werner (WS), Bloom (BS), and Rothmund-Thomson syndromes (RTS), respectively [3,4,5]. The functions of RecQ helicases in genome maintenance suppress cancer and perhaps other phenotypes associated with aging
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