RecQ helicases maintain
chromosome stability by resolving several highly specific
DNA structures. BLM, the
protein mutated in
Bloom's syndrome, is a member of the
RecQ helicase family, and possesses both
DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal
DNA, the natural nuclear substrate for the
enzyme. We generated
a DNA template including a strong
nucleosome-positioning sequence flanked by forked
DNA, which is reportedly one of the preferred
DNA substrates for BLM. BLM did not possess detectable unwinding activity toward the forked
DNA substrate. However, the truncated BLM, lacking annealing activity, unwound it partially. In the presence of the
single-stranded DNA-binding protein RPA, the unwinding activity of both the full-length and the truncated BLMs was promoted. Next, the
histone octamer was reconstituted onto the forked
DNA to generate a forked mononucleosome. Full-length BLM did not unwind the nucleosomal
DNA, but truncated BLM unwound it partially. The unwinding activity for the mononucleosome was not promoted dramatically with RPA. These results indicate that full-length BLM may require additional factors to unwind nucleosomal
DNA in vivo, and that RPA is, on its own, unable to perform this auxiliary function.