DNA double-strand breaks (DSBs) are highly toxic and must usually be accurately repaired to counteract the threat of oncogenic mutations. However some specialist tissues actually rely on mutagenic DSB repair as a means by which genetic material can be transferred between loci to create genetic diversity. To cope with this intrinsic discrepancy in desired DNA repair outcome between different tissues and cellular contexts, a complex regulatory system has evolved to maintain the right equilibrium between competing DNA repair pathways, ensuring DSBs are appropriately resolved. Failure of this system is known to be a key driver of hereditary and spontaneously occurring cancers. It also contributes to the pathologies of multiple genetic syndromes such as Ataxia Telangiectasia, Nijmegen Breakage Syndrome, and Severe Combined Immunodeficiency (SCID), whose pathologies include developmental defects, infertility, immunodeficiency and cancer predisposition.
In recent work from our laboratory, we have shown misregulation of this system to link the DNA repair mechanisms that create immunodiversity to those that drive cancer. Specifically, a chromatin component of the non-homologous end joining (NHEJ) DSB repair pathway that is vital for mediating antibody gene rearrangements in lymphocytes, is additionally responsible for the genomic instability that drives tumourigenesis in patients harbouring mutations in the BRCA breast cancer tumour suppressor genes. Moreover, we have also shown that the repair activities of this branch of the NHEJ pathway can be harnessed in therapeutic treatment regimes to selectively kill cancer cells.
The aim of this project is to define the molecular mechanisms that regulate the choice between the two major DSB repair pathways, and determine their contributions to tumour suppression and oncogenesis in humans. To achieve this, biochemical approaches will be employed together with newly developed genetic screening systems to determine how the BRCA1 protein functions in counteracting mutagenic DSB repair events, and additionally identify the molecular mechanisms responsible for triggering BRCA-associated cancers.
Funding: Research in the Chapman lab is supported by grants from Cancer Research UK, the Wellcome Trust, the Medical Research Council, and the European Commission via Horizon 2020.
The successful applicant will receive training in a wide range of modern molecular biology, biochemistry and cell biology techniques, using targeted CRISPR genome editing, and genome-wide CRISPR screening approaches to model protein function and reveal the important components of the core DSB repair pathways.
Housed at the Wellcome Trust Centre for Human Genetics, the candidate will profit from state-of-the-art core facilities that include high-throughput genomics (Next Generation and single-cell sequencing), bioinformatics, transgenics, confocal and super-resolution microscopy. The candidate will also benefit from the complementary work of other teams working in the institute, and interactions with the active genome stability community across the university.
It is expected that the applicant will also gain a broad range of other skills during the course of the project. This includes time and project management, supervision of junior staff members, use of scientific web resources, and scientific writing and reviewing. There will also be opportunities to travel to international conferences to present results obtained during the project.
Project reference number: 658
|Dr Ross Chapman||Wellcome Trust Centre for Human Genetics||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Professor Ian Tomlinson||Wellcome Trust Centre for Human Genetics||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRemail@example.com|
Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells. Hide abstract
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5' end resection, but how this is achieved remains uncertain. Here we report that Rif1(-/-) mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination. Hide abstract
DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability. Hide abstract
Following irradiation, numerous DNA-damage-responsive proteins rapidly redistribute into microscopically visible subnuclear aggregates, termed ionising-radiation-induced foci (IRIF). How the enrichment of proteins on damaged chromatin actually relates to DNA repair remains unclear. Here, we use super-resolution microscopy to examine the spatial distribution of BRCA1 and 53BP1 proteins within single IRIF at subdiffraction-limit resolution, yielding an unprecedented increase in detail that was not previously apparent by conventional microscopy. Consistent with a role for 53BP1 in promoting DNA double-strand break repair by non-homologous end joining, 53BP1 enrichment in IRIF is most prominent in the G0/G1 cell cycle phases, where it is enriched in dense globular structures. By contrast, as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, leading to an overall reduction of 53BP1 occupancy at DNA damage sites. Our data suggest that the BRCA1-associated IRIF core corresponds to chromatin regions associated with repair by homologous recombination, and the enrichment of BRCA1 in IRIF represents a temporal switch in the DNA repair program. We propose that BRCA1 antagonises 53BP1-dependent DNA repair in S phase by inhibiting its interaction with chromatin proximal to damage sites. Furthermore, the genomic instability exhibited by BRCA1-deficient cells might result from a failure to efficiently exclude 53BP1 from such regions during S phase. Hide abstract
The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response. Hide abstract
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination. Hide abstract