Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair

Nicholas L Adkins,Sarah G Swygert,Parminder Kaur,Hengyao Niu,Sergei A Grigoryev,Patrick Sung,Hong Wang,Craig L Peterson

doi:10.1074/jbc.m117.776369

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 after in vitro chromatin 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-enhanced electrostatic force microscopy). The reconstituted ssNucs are homogenous and relatively stable, and DREEM reveals ssDNA wrapping around histones. We also find that histone octamers are easily transferred in trans from 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.

Highlights

Leading to numerous diseases [1,2,3]
Our results support the view that ssNucs are a hallmark of double strand breaks (DSBs) chromatin that has been processed for homologous recombination (HR), and we suggest that these structures may present novel opportunities for histone posttranslational modifications to contribute to the cellular response to DNA damage
Histones Remain Associated with Single-stranded DNA after in Vitro DNA Resection—Previously, we reported that nucleosomes are not a barrier for DNA resection by the Sgs1-Dna2 machinery if sufficient free DNA is located adjacent to a positioned nucleosome [19]

Summary

Introduction

Leading to numerous diseases [1,2,3]. There are two main pathways for DNA DSB repair: error-prone, non-homologous endjoining and relatively error-free, homologous recombination (HR)2 [4]. These single-stranded histone complexes, which we term ssNucs, display structural features similar to canonical nucleosomes with ssDNA wrapping around histone proteins, as shown in DREEM imaging. To characterize the composition of single-stranded histone complexes, we reconstituted ssDNA and dsDNA complexes with biotin-labeled DNA, and histone stoichiometry was analyzed following magnetic bead capture by SDS-PAGE (Fig. 2D).

Results

Conclusion