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Maintenance of genome stability is of paramount importance for the survival of an organism. However, genomic integrity is constantly being challenged by various endogenous and exogenous processes that damage DNA. Therefore, cells are heavily reliant on DNA repair pathways that have evolved to deal with every type of genotoxic insult that threatens to compromise genome stability. Notably, inherited mutations in genes encoding proteins involved in these protective pathways trigger the onset of disease that is driven by chromosome instability e.g. neurodevelopmental abnormalities, neurodegeneration, premature ageing, immunodeficiency and cancer development. The ability of cells to regulate the recruitment of specific DNA repair proteins to sites of DNA damage is extremely complex but is primarily mediated by protein post-translational modifications (PTMs). Ubiquitylation is one such PTM, which controls genome stability by regulating protein localisation, protein turnover, protein-protein interactions and intra-cellular signalling. Over the past two decades, numerous ubiquitin (Ub) E3 ligases have been identified to play a crucial role not only in the initiation of DNA replication and DNA damage repair but also in the efficient termination of these processes. In this review, we discuss our current understanding of how different Ub E3 ligases (RNF168, TRAIP, HUWE1, TRIP12, FANCL, BRCA1, RFWD3) function to regulate DNA repair and replication and the pathological consequences arising from inheriting deleterious mutations that compromise the Ub-dependent DNA damage response., (© 2024 The Author(s).)

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.

The development of the human central nervous system is a complex process involving highly coordinated periods of neuronal proliferation, migration and differentiation. Disruptions in these neurodevelopmental processes can result in microcephaly, a neuropathological disorder characterized by a reduction in skull circumference and total brain volume, whereas a failure to maintain neuronal health in the adult brain can lead to progressive neurodegeneration. Defects in the cellular pathways that detect and repair DNA damage are a common cause of both these neuropathologies and are associated with a growing number of hereditary human disorders. In particular, defects in the repair of DNA single strand breaks, one of the most commonly occurring types of DNA lesion, have been associated with three neuropathological diseases: ataxia oculomotor apraxia 1, spinocerebellar ataxia with neuronal neuropathy 1 and microcephaly, early-onset, intractable seizures and developmental delay. A striking similarity between these three human diseases is that they are all caused by mutations in DNA end processing factors, suggesting that a particularly crucial stage of DNA single strand break repair is the repair of breaks with 'damaged' termini. Additionally all three disorders lack any extraneurological symptoms, such as immunodeficiency and cancer predisposition, which are typically found in other human diseases associated with defective DNA repair. However despite these similarities, two of these disorders present with progressive cerebellar degeneration, whereas the third presents with severe microcephaly. This review discusses the molecular defects behind these disorders and presents several hypotheses based on current literature on a number of important questions, in particular, how do mutations in different end processing factors within the same DNA repair pathway lead to such different neuropathologies?

DNA double-strand break (DSB) repair is an essential cellular process required to maintain genomic integrity in the face of potentially lethal genetic damage. Failure to repair a DSB can trigger cell death, whereas misrepair of the break can lead to the generation of chromosomal translocations, which is a known causative event in the development or progression of cancer. DSBs can be induced following exposure to certain exogenous agents, such as ionising radiation or radiomimetic chemicals, as well as occurring naturally as intermediates of normal physiological processes, in particular during B and T cell antigen receptor assembly. Human syndromes with deficiencies in DSB repair commonly exhibit immunodeficiency, highlighting the critical nature of this pathway for development and maturation of the immune system. In this article we review the different pathways utilized by the cell to repair DSBs and how an inherited defect in some of the genes that are critical regulators of this process can be the underlying cause of human disorders associated with genome instability and immune system dysfunction. We focus on a newly described human immunodeficiency disorder called radiosensitivity, immunodeficiency dysmorphic features and learning difficulties (RIDDLE) syndrome, with particular reference to the function of the defective gene, RNF168. We also consider the implications of this finding on the mechanisms controlling development of the immune system.

Through the action of multiple sensors, mediators, and effectors, the DNA damage response (DDR) orchestrates the repair of DNA damage to ensure maintenance of genomic integrity. Recently, in addition to phosphorylation, other post-translational modifications such as ubiquitylation and SUMOylation have emerged as important regulators of the DDR network. Two recent papers highlight the importance of SUMO modifications of proteins that execute the response to DNA damage., ((c) 2010 Elsevier B.V. All rights reserved.)

The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes.

DNA double-strand breaks (DSBs) trigger ATM (ataxia telangiectasia mutated) signalling and elicit genomic rearrangements and chromosomal fragmentation if misrepaired or unrepaired. Although most DSB repair is ATM-independent, approximately 15% of ionizing radiation (IR)-induced breaks persist in the absence of ATM-signalling. 53BP1 (p53-binding protein 1) facilitates ATM-dependent DSB repair but is largely dispensable for ATM activation or checkpoint arrest. ATM promotes DSB repair within heterochromatin by phosphorylating KAP-1 (KRAB-associated protein 1, also known as TIF1beta, TRIM28 or KRIP-1; ref. 2). Here, we show that the ATM signalling mediator proteins MDC1, RNF8, RNF168 and 53BP1 are also required for heterochromatic DSB repair. Although KAP-1 phosphorylation is critical for 53BP1-mediated repair, overall phosphorylated KAP-1 (pKAP-1) levels are only modestly affected by 53BP1 loss. pKAP-1 is transiently pan-nuclear but also forms foci overlapping with gammaH2AX in heterochromatin. Cells that do not form 53BP1 foci, including human RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome cells, fail to form pKAP-1 foci. 53BP1 amplifies Mre11-NBS1 accumulation at late-repairing DSBs, concentrating active ATM and leading to robust, localized pKAP-1. We propose that ionizing-radiation induced foci (IRIF) spatially concentrate ATM activity to promote localized alterations in regions of chromatin otherwise inhibitory to repair.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease involving more than one molecular mechanism that leads to the transformation of CD5(+) B cells at either the pregerminal or postgerminal center stage of differentiation. It was previously demonstrated that ataxia telangiectasia mutated (ATM) gene mutations can occur in B-CLL and cause a defect in the p53 pathway. Here the role of ATM mutations in the pathogenesis of B-CLL is addressed. Of 50 B-CLL tumors with fully analyzed ATM and TP53, 16 had ATM mutations. Six of 50 B-CLLs showed mutations in TP53 and the remaining 28 tumors had wild-type ATM or TP53. No tumor had both ATM and TP53 mutations. Remarkably, all 16 ATM mutant B-CLLs showed the absence of somatic variable region heavy chain hypermutation indicating a pregerminal center cell origin and a common pathogenesis for these tumors. Furthermore, in 5 of the 16 B-CLLs, ATM mutation preceded the transformation stage of differentiation. At the cellular level, ATM mutant tumors exhibited a deficient ATM-dependent p53 response to gamma irradiation, failure to up-regulate TRAIL-R2, a downstream target that links irradiation-induced p53 response with apoptosis, and an inability to repair induced chromosome breaks. Mantle cell lymphoma (MCL) is also of pregerminal center origin and ATM mutations are frequent in this malignancy. It is concluded that ATM is likely to play an important role at the pregerminal center stage and a model is proposed where loss of ATM function during B-cell ontogeny drives B-CLL tumorigenesis in pregerminal B cells by a dual defect in p53 damage response and repair of chromosome breaks.

The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4–Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4–Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S. [ABSTRACT FROM AUTHOR]

Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele. [ABSTRACT FROM AUTHOR]

Abstract: Ataxia-telangiectasia (A–T) has for a long time stood apart from most other human neurodegenerative syndromes by the characteristic failure of cells derived from these patients to properly repair DNA damage-induced by ionizing radiation. The discovery of mutations in the ATM gene as being the underlying cause for A–T and the demonstration that the ATM protein functions as a DNA damage-responsive kinase has defined current research focusing on decoding how the cell responds to genotoxic stress. Yet, despite significant advances in delineating the cellular DNA damage response pathways coordinated by ATM, very little headway has been made toward understanding how loss of ATM leads to progressive cerebellar ataxia and whether this can be attributed to an underlying defect in DNA double strand break repair (DSBR). Since its identification, A–T has been used as the archetypal model for how a deficiency in DNA repair affects both the development and maintenance of the nervous and immune systems in humans as well as contributing to the process of tumourigenesis. However, following the growing availability and cost effectiveness of next generation sequencing technologies, the increasing recognition of novel human disorders associated with abnormal DNA repair has demonstrated that the neuropathology typified by A–T is an ‘exception’ rather than the ‘rule’. As a consequence, this throws into doubt the longstanding hypothesis that the neurodegeneration seen in A–T is due to the progressive loss of damaged neurons that have acquired toxic levels of unrepaired DNA lesions over time. Therefore, this review aims to address the question: Is defective DNA double strand break repair an underlying cause of neurodegeneration? [Copyright &y& Elsevier]

The development of the human central nervous system is a complex process involving highly coordinated periods of neuronal proliferation, migration and differentiation. Disruptions in these neurodevelopmental processes can result in microcephaly, a neuropathological disorder characterized by a reduction in skull circumference and total brain volume, whereas a failure to maintain neuronal health in the adult brain can lead to progressive neurodegeneration. Defects in the cellular pathways that detect and repair DNA damage are a common cause of both these neuropathologies and are associated with a growing number of hereditary human disorders. In particular, defects in the repair of DNA single strand breaks, one of the most commonly occurring types of DNA lesion, have been associated with three neuropathological diseases: ataxia oculomotor apraxia 1, spinocerebellar ataxia with neuronal neuropathy 1 and microcephaly, early-onset, intractable seizures and developmental delay. A striking similarity between these three human diseases is that they are all caused by mutations in DNA end processing factors, suggesting that a particularly crucial stage of DNA single strand break repair is the repair of breaks with ‘damaged’ termini. Additionally all three disorders lack any extraneurological symptoms, such as immunodeficiency and cancer predisposition, which are typically found in other human diseases associated with defective DNA repair. However despite these similarities, two of these disorders present with progressive cerebellar degeneration, whereas the third presents with severe microcephaly. This review discusses the molecular defects behind these disorders and presents several hypotheses based on current literature on a number of important questions, in particular, how do mutations in different end processing factors within the same DNA repair pathway lead to such different neuropathologies?. [ABSTRACT FROM PUBLISHER]

Abstract: Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes. [Copyright &y& Elsevier]

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability. [ABSTRACT FROM AUTHOR]

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with ?-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal. [ABSTRACT FROM AUTHOR]

• Ogg1-null mouse embryonic fibroblasts (MEFs) are more sensitive to depletion of glutathione. • Ogg1-null MEFs possess increased basal levels of intracellular reactive oxygen species. • Ogg1-null MEFs do not accumulate genomic 8-oxoG lesions. • Glutathione depletion induces apoptosis in Ogg1-null MEFs. Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (m Ogg1 −/− MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in m Ogg1 −/− MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in m Ogg1 -/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1 −/− MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in m Ogg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into m Ogg1 −/− MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in m Ogg1 −/− MEFs is independent of DNA damage and OGG1-initiated DNA repair. [ABSTRACT FROM AUTHOR]

Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication. IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection. [ABSTRACT FROM AUTHOR]

The 53BP1-RIF1-shieldin pathway maintains genome stability by suppressing nucleolytic degradation of DNA ends at double-strand breaks (DSBs). Although RIF1 interacts with damaged chromatin via phospho-53BP1 and facilitates recruitment of the shieldin complex to DSBs, it is unclear whether other regulatory cues contribute to this response. Here, we implicate methylation of histone H3 at lysine 4 by SETD1A-BOD1L in the recruitment of RIF1 to DSBs. Compromising SETD1A or BOD1L expression or deregulating H3K4 methylation allows uncontrolled resection of DNA ends, impairs end-joining of dysfunctional telomeres, and abrogates class switch recombination. Moreover, defects in RIF1 localization to DSBs are evident in patient cells bearing loss-of-function mutations in SETD1A. Loss of SETD1A-dependent RIF1 recruitment in BRCA1- deficient cells restores homologous recombination and leads to resistance to poly(ADP-ribose)polymerase inhibition, reinforcing the clinical relevance of these observations. Mechanistically, RIF1 binds directly to methylated H3K4, facilitating its recruitment to, or stabilization at, DSBs. [Display omitted] • BOD1L, SET1A, and H3K4me3 promote RIF1 accumulation at DNA break sites • SETD1A-dependent H3K4 methylation promotes end-joining and suppresses end-resection • Perturbing SETD1A confers resistance to PARP inhibitors in BRCA1-deficient cells • RIF1 binds directly to methylated H3K4 Bayley et al. identify histone H3K4 methylation by SETD1A as vital for DNA repair, by promoting RIF1 accumulation at damaged sites. Deficiencies in H3 methylation or the SETD1A-BOD1L complex impairs end-joining of broken DNA ends, abrogates class switch recombination, promotes uncontrolled end-resection, and compromises the efficacy of PARP inhibitors. [ABSTRACT FROM AUTHOR]

Summary: DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs. [Copyright &y& Elsevier]

Summary Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization. [ABSTRACT FROM AUTHOR]