Does BLM helicase unwind nucleosomal DNA?
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.
Fujimoto
Satoru
S
Department of Molecular Biology, College of Agriculture, University of Wyoming, 1000 East University Ave., Laramie, WY 82071, USA.
Tomschik
Miroslav
M
Zlatanova
Jordanka
J
D001483:Base Sequence; D001816:Bloom Syndrome / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000235:genetics* / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism
D009707:Nucleosomes; D051716:Replication Protein A; D004247:DNA; C097151:Bloom syndrome protein; D053484:RecQ Helicases