E3 ligases:a ubiquitous link between DNA repair, DNA replication and human disease

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.</p

DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair.

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.T.O. and T.L.B. are supported by the Wellcome Trust. The Jackson lab is funded by Cancer Research UK (CRUK) program grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010- 259893 (DDResponse). Core infrastructure funding to the Jackson lab is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. V.M.D. is a CRUK Career Development Fellow. The Draviam lab is funded by a CRUK CDA (C28598/A9787).This is the accepted manuscript version. The final version is available from AAAS at http://www.sciencemag.org/content/347/6218/185.full

MDC1 PST-repeat region promotes histone H2AX-independent chromatin association and DNA damage tolerance

Abstract: Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX

Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability

Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

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

Research data supporting "Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair"

Complete set of graphs for gammaH2AX, 53BP1 and ubiquitin (FK2 antibody) foci/cell kinetics in response to ionising radiation (IR)- and mock-treated human U2OS cells treated with siRNAs targeting ubiquitin E2s (screening module 1; refers to Fig. 1). Complete set of DDR signalling immunoblots, generated from whole cell extracts of IR-, camptothecin- or mock-treated human U2OS cells treated with siRNAs targeting ubiquitin E2s in screening module 3 (refers to Fig. 3 and Supplementary Fig. 2).This work was supported by the European Community Seventh Framework Programme [grant number HEALTH-F2-2010-259893], the European Research Council (DDREAM), CRUK [grant number C6946/A14492], Wellcome Trust [grant number WT092096], the German Research Foundation (DFG) [BE 5342/1-1], and the Return-to-Europe Fellowship (FEBS)

Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ncb3260Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumourigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2- enzymes for impacts on cellular DSB responses. Applying a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3-ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.Alex Sossick, Nicola Lawrence and Richard Butler. Research in the S.P.J. lab is funded by Cancer Research UK Program Grant C6/A11224, the European Research Council (DDREAM), the European Community Seventh Framework Programme grant agreement no. HEALTH-F2- 2010-259893 (DDResponse). Core infrastructure funding was provided by Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096. S.P.J. receives a salary from the University of Cambridge, supplemented by Cancer Research UK. C.K.S. was funded by a FEBS Return-to-Europe fellowship. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1)

Cancer-Associated SF3B1 Mutations Confer a BRCA-Like Cellular Phenotype and Synthetic Lethality to PARP Inhibitors

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionising radiation and a variety of chemotherapeutic agents, including PARP inhibitors. Additionally, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation and defective replication fork restart. Taken together, these data suggest that tumour-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA damaging agents and PARP inhibitors, which can be exploited therapeutically