HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: implications for Fanconi anemia and breast cancer susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.     

DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.     

Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites?of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR. PAPERFLICK:     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.     

The 19S proteasome subunit Rpn7 stabilizes DNA damage foci upon genotoxic insult

The DNA damage response (DDR) orchestrates the recruitment of repair proteins at sites of damage and arrests cell-cycle progression until completion of repair. Upon irreparable damage, DNA damage foci persist (long-lived foci) and this is believed to induce cellular senescence. The resolution of DNA damage foci has previously been shown to depend on proteasomal degradation and various proteasome subunits have been implicated in the DDR. In this study, we aimed to analyze the possible distinct roles of individual proteasome subunits in the DDR. We show that specific 19S subunits respond to DNA damage by increased protein levels and nuclear translocation. Importantly, two 19S subunits, Rpn7 and Rpn11, colocalize with DNA damage foci over their whole lifespan. Although silencing of Rpn11 does not affect foci stability and lifespan, silencing of Rpn7 promotes faster resolution of DNA damage foci following genotoxic insult. For the first time, we provide evidence that Rpn7 silencing specifically decreases the frequencies of long-lived DNA damage foci without, however, affecting the repair rate of short-lived foci. Therefore, we propose that interaction of Rpn7 with DDR foci in situ mediates the protection of DNA damage foci from premature resolution. We suggest that this interaction is involved in enabling cellular senescence following genotoxic insult.     

Spatiotemporal regulation of posttranslational modifications in the DNA damage response

A timely and accurate cellular response to DNA damage requires tight regulation of the action of DNA damage response (DDR) proteins at lesions. A multitude of posttranslational modifications (PTMs) of chromatin and chromatin‐associated proteins coordinates the recruitment of critical proteins that dictate the appropriate DNA repair pathway and enable the actual repair of lesions. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP‐ribosyl)ation, acetylation, and methylation are among the DNA damage‐induced PTMs that have taken center stage as important DDR regulators. Redundant and multivalent interactions of DDR proteins with PTMs may not only be a means to facilitate efficient relocalization, but also a feature that allows high temporal and spatial resolution of protein recruitment to, and extraction from, DNA damage sites. In this review, we will focus on the complex interplay between such PTMs, and discuss the importance of their interconnectivity in coding DNA lesions and maintaining the integrity of the genome.     

53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response

TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of γ-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)- and the ataxia telangiectasia and Rad3 related (ATR)-mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes.     

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage

Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.     

Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.     

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)—a highly cytotoxic DNA lesion—activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.     

ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.