The chromatin response to DNA breaks: leaving a mark on genome integrity.

Genetic, biochemical, and cellular studies have uncovered many of the molecular mechanisms underlying the signaling and repair of chromosomal DNA breaks. However, efficient repair of DNA damage is complicated in that genomic DNA is packaged, through histone and nonhistone proteins, into chromatin. The DNA repair machinery has to overcome this physical barrier to gain access to damaged DNA and repair DNA lesions. Posttranslational modifications of chromatin as well as ATP-dependent chromatin remodeling factors help to overcome this barrier and facilitate access to damaged DNA by altering chromatin structure at sites of DNA damage. Here we review and discuss our current knowledge of and recent advances in chromatin changes induced by chromosome breakage in mammalian cells and their implications for genome stability and human disease.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling.

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of γH2AX into damaged chromatin, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168 ubiquitin cascade. Moreover, SMARCA5 was found to regulate the ubiquitin response by promoting RNF168 accumulation at DSBs, which subsequently facilitates efficient ubiquitin conjugation and BRCA1 assembly. Underlining the importance of these findings, we show that SMARCA5 depletion renders cells sensitive to IR and results in DSB repair defects. Our study unveils a functional link between DNA damage-induced poly(ADP-ribosyl)ation, SMARCA5-mediated chromatin remodeling and RNF168-dependent signaling and repair of DSBs.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness.

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.

Cells respond to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin-remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.