title: Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair.
abstract: Repair of DNA double strand breaks (DSBs) is key for maintenance of genome integrity. When DSBs are repaired by homologous recombination, DNA ends can undergo extensive processing, producing long stretches of single-stranded DNA (ssDNA).In vivo, DSB processing occurs in the context of chromatin, and studies indicate that histones may remain associated with processed DSBs. Here we demonstrate that histones are not evicted from ssDNA afterin vitrochromatin resection. In addition, we reconstitute histone-ssDNA complexes (termed ssNucs) with ssDNA and recombinant histones and analyze these particles by a combination of native gel electrophoresis, sedimentation velocity, electron microscopy, and a recently developed electrostatic force microscopy technique, DREEM (dual-resonance frequency-enhancedelectrostatic forcemicroscopy). The reconstituted ssNucs are homogenous and relatively stable, and DREEM reveals ssDNA wrapping around histones. We also find that histone octamers are easily transferred intransfrom ssNucs to either double-stranded DNA or ssDNA. Furthermore, the Fun30 remodeling enzyme, which has been implicated in DNA repair, binds ssNucs preferentially over nucleosomes, and ssNucs are effective at activating Fun30 ATPase activity. Our results indicate that ssNucs may be a hallmark of processes that generate ssDNA, and that posttranslational modification of ssNucs may generate novel signaling platforms involved in genome stability.
authors.affiliation: From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Nicholas L
authors.initials: NL
authors.lastname: Adkins
authors.affiliation: From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605.
authors.forename: Sarah G
authors.initials: SG
authors.lastname: Swygert
authors.affiliation: the Department of Physics.|Center for Human Health and the Environment, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Parminder
authors.initials: P
authors.lastname: Kaur
authors.affiliation: the Department Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, and.
authors.forename: Hengyao
authors.initials: H
authors.lastname: Niu
authors.affiliation: the Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Grigoryev
authors.affiliation: the Department Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, and.
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
authors.affiliation: the Department of Physics.|Center for Human Health and the Environment, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, craig.peterson@umassmed.edu.
authors.forename: Craig L
authors.initials: CL
authors.lastname: Peterson
mesh_terms: D042002:Chromatin Assembly and Disassembly / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D042822:Genomic Instability; D006657:Histones / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; C074000:FUN30 protein, S cerevisiae; D006657:Histones; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
keywords: DNA repair; DSB processing; analytical ultracentrifugation; atomic force microscopy (AFM); chromatin remodeling; nucleosome; ssDNA