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

Abstract

Edited by John M. DenuRepair 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.

Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein

“…Topographic and DREEM images are obtained simultaneously by mechanically vibrating the AFM cantilever near the fundamental resonance (ω 1 ), while applying a static voltage (V DC ) and a modulated bias voltage (V AC at the first overtone ω 2 ) between the AFM cantilever and mica substrate. In DREEM imaging, both free proteins and DNA show a decrease in phase compared to the mica surface, but proteins show a greater contrast than DNA, which permits the identification of DNA paths in protein–DNA complexes ( 32–34 , 48 ).…”

“…All images were captured at a scan rate of 1–2 Hz and a resolution of 512 × 512 pixels. DREEM images were collected as described previously ( 32–34 , 48 ). Briefly, the AFM cantilever and the bottom of the mica substrate were coated with a thin layer of colloidal liquid silver (Ted Pella Inc.).…”

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation

“…Although BRCA1-directed PALB2 recruitment to ssDNA is critical for RAD51-dependent HR (Sy et al, 2009;Zhang et al, 2009aZhang et al, , 2009b, the physiological relevance of RNF168mediated PALB2 chromatin loading has remained unclear (Luijsterburg et al, 2017). Based on our finding that cells become reliant on the RNF168-dependent pathway when BRCA1 protein level or its interaction with PALB2 is reduced by 50%, we suggest that ubiquitin polymers on histones assembled on ssDNA after end resection (Adkins et al, 2017;Huang et al, 2018) or surrounding the processed ssDNA compartment provide a backup mechanism to load RAD51 ( Figure 6C). In BRCA1/53BP1-deficient cells, such a ubiquitin platform becomes essential to restore HR and viability.…”

The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes

“…Biochemically, this remodeler has been best characterized in budding yeast. Fun30 is capable of binding chromatin and DNA in vitro with a preference for single-stranded chromatin and exhibits activity in ATP-dependent chromatin remodeling assays (19,23,24). A homozygous mutation of this remodeler in the mouse results in growth retardation, prenatal and perinatal lethality, reduced fertility, and skeletal abnormali-ties (25).…”