title: Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase.
abstract: There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3' single-stranded gap and a 5' double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Life Science and Technology, Northwest A & F University, Yangling, Shaanxi 712100, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Qin
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Jing-Hua
authors.initials: JH
authors.lastname: Li
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Lu
authors.affiliation: College of Life Science and Technology, Northwest A & F University, Yangling, Shaanxi 712100, China.
authors.forename: Ke-Yu
authors.initials: KY
authors.lastname: Lu
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Da-Guan
authors.initials: DG
authors.lastname: Nong
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Shuo-Xing
authors.initials: SX
authors.lastname: Dou
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China xch@iphy.ac.cn.
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Xu
authors.affiliation: College of Life Science and Technology, Northwest A & F University, Yangling, Shaanxi 712100, China Laboratoire de Biologie et PharmacologieAppliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61 Avenue du Président Wilson, 94235 Cachan, France xxi01@ens-cachan.fr.
authors.forename: Xu-Guang
authors.initials: XG
authors.lastname: Xi
authors.affiliation: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
mesh_terms: D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D053484:RecQ Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C097151:Bloom syndrome protein; D053484:RecQ Helicases