title: BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation.
abstract: Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.
authors.affiliation: Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Jaya G
authors.initials: JG
authors.lastname: Yodh
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Stevens
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Kanagaraj
authors.forename: Pavel
authors.initials: P
authors.lastname: Janscak
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D001816:Bloom Syndrome / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics; D004260:DNA Repair; D004261:DNA Replication; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D053484:RecQ Helicases / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D004247:DNA; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases