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.
Highlights
Family discovered in Escherichia coli [1]
Mutations in the RECQ1 and RECQ5 genes may be responsible for additional cancer predisposition disorders that are distinct from Rothmund-Thomson syndrome, Bloom’s syndrome, and Werner’s syndrome, but this remains to be proven
Collectively, our findings show that RECQ1 has a distinct substrate specificity compared with BLM, providing a strong indication that these helicases are likely to perform nonoverlapping functions in cells
Summary
Proteins—Recombinant His6-tagged RECQ1 and BLM were expressed and purified following previously described procedures [28, 29]. For the experiments with the untagged RECQ1, the His tag sequence was removed by digestion with thrombin (1:500 ratio) for 3 h at 4 °C in a buffer of 20 mM Tris-HCl, pH 7.4, 150 mM KCl, 5 mM -mercaptoethanol. The experiments with untagged BLM were performed with a truncated variant of the protein, BLM-(642–1290), expressed and purified from E. coli following previously described procedures [30]. [␥32P]ATP-labeled oligonucleotides were annealed to a 1.4-fold excess of the unlabeled complementary strands in annealing buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl) by heating at 95 °C for 6 min and cooling slowly to room temperature.
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