The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities

Abstract

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.

Structure of the human RECQ1 helicase reveals a putative strand-separation pin

“…1 C and D). This confirms the previous observations that higher-order oligomers are required for the strand-annealing activity of RECQ1 and suggests that these oligomers are also necessary for the HJ resolution activity of RECQ1 (22).…”

“…In parallel, the truncated protein lacks HJ unwinding activity, as well as a DNA-annealing activity. This confirms the previous observations that the higher-oligomeric forms of RECQ1 are involved in DNA strand annealing (22,23) and suggest that they might be also be required for the disruption of HJ structures (22). In contrast, the truncated RECQ1 T1 protein is as active as the full-length protein in unwinding forked duplexes, confirming the earlier observation that a monomer or dimer of RECQ1 can perform this activity (22,23).…”

“…There are several reports of differences in substrate specificity between RecQ family members. For example, differences between fulllength RECQ1 and BLM suggest that there must be some key structural features that distinguish the catalytic domains of these 2 enzymes (22). The substrate specificity of RECQ1 is also distinct from that of E. coli RecQ because, for example, the bacterial enzyme can unwind blunt-ended duplexes and Gquadruplex DNA, which cannot be resolved by RECQ1 (41,42).…”

“…Even when acting on the same substrates, they may do so by using different mechanisms (22). In addition to unwinding DNA, some RecQ helicases are also able to promote the annealing of complementary ssDNA molecules in an ATP-independent fashion (23)(24)(25)(26)(27)(28).…”

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

“…Most RecQ enzymes, including EcRecQ, unwind a very broad array of substrates that include duplex, triplex, quadruplex, and branched DNA (3). Human RecQ1, which unwinds Holliday junction but not triplex or quadruplex DNA, is a rare exception in its narrow substrate specificity (13). The mechanisms that confer functional diversity in most, but not all, RecQ enzymes remain unknown.…”