Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

Abstract

Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction

“…We also take into consideration the probable role of post-translational modification on the helicase activity of RECQL4. Unlike our study, most previous reports used bacterial expression system for purification (6,7). Furthermore, another study using baculovirus system purified human RECQL4 also showed relatively weaker helicase activity (5).…”

“…However, robust ATP-dependent helicase activity for human RECQL4 has not been demonstrated for substrates with double-stranded regions longer than 22 bp (3,(5)(6)(7)30). Typically demonstration of strand exchange activity has been reported for substrates with longer double-stranded regions in the presence of excess single-stranded traps.…”

“…Purification of Human RECQL4 and Helicase-dead K508N-RECQL4 Using a Baculovirus System-Purification of human RECQL4 using bacterial and baculovirus expression systems has been reported previously (3,(5)(6)(7). We used a baculovirus expression system for purification of RECQL4 and helicasedead K508N-RECQL4 mutant.…”

“…Initial reports suggested RECQL4 lacked of helicase activity (3), but annealing, strand exchange and helicase activities of RECQL4 were later demonstrated (4 -7). Human RECQL4 was shown to unwind up to 22-bp double-stranded regions through its helicase activity (6,7). ATPase and annealing activities of human RECQL4 have also been extensively studied (7,8).…”

“…Human RECQL4 was shown to unwind up to 22-bp double-stranded regions through its helicase activity (6, 7). ATPase and annealing activities of human RECQL4 have also been extensively studied (7,8). Robust helicase and annealing activities on substrates of various lengths have been reported for Drosophila melanogaster RecQ4 (DmRecQ4) (4).…”