title: RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein.
abstract: Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg(2+) concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.
authors: Department of Chemical Engineering, University of Michigan, 3074 H.H. Dow, 2300 Hayward Street, Ann Arbor, MI 48109-2136, USA.
authors: Wei-Xian
authors: WX
authors: Shi
authors: Ronald G
authors: RG
authors: Larson
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D008274:Magnesium