title: Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates.
abstract: Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.
authors.affiliation: 3Department of Biochemistry, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary. kovacsm@elte.hu.
authors.forename: Máté
authors.initials: M
authors.lastname: Gyimesi
authors.forename: Ricardo H
authors.initials: RH
authors.lastname: Pires
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.forename: Kata
authors.initials: K
authors.lastname: Sarlós
authors.forename: Zsuzsa S
authors.initials: ZS
authors.lastname: Kocsis
authors.forename: Károly
authors.initials: K
authors.lastname: Módos
authors.forename: Miklós S Z
authors.initials: MS
authors.lastname: Kellermayer
authors.forename: Mihály
authors.initials: M
authors.lastname: Kovács
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D059765:Homologous Recombination; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D053484:RecQ Helicases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; C097151:Bloom syndrome protein; D053484:RecQ Helicases
keywords: DNA repair; atomic force microscopy; electron microscopy; oligomerization; structure