Dr Ross Chapman
| Research Area: | Cell and Molecular Biology |
|---|---|
| Technology Exchange: | Cellular immunology, Microscopy (Confocal) and Protein interaction |
| Scientific Themes: | Cancer Biology and Physiology, Cellular & Molecular Biology |
| Keywords: | Genome Stability, DNA double-strand break repair, Chromatin and Epigenetics |
| Web Links: |
BRCA1 and 53BP1 regulate DNA double strand break repair pathway choice. BRCA1 (green) and 53BP1 ...
Chromatin-dependent regulation of DNA repair. A dynamic competition between DNA repair factors and ...
RIF1-deficiency in mice results in aberrant nucleolytic processing of DSBs during immunoglobulin ...
Chromatin and Genome Integrity
We aim to discover the function of molecular events propagated in chromatin upon DNA damage detection, and question why defects in these events manifest in immune-deficiency and cancer in humans.
DNA double-strand breaks (DSBs) are a highly toxic form of DNA damage, which if not properly repaired can result in mutations and genomic translocations. However, DSBs are also required during the specialized recombination events that generate diversity in our immune systems. Accordingly, cellular responses to DSBs are tightly regulated in a cell type and cell cycle dependent manner.
Two core regulators of DNA repair pathway choice are the BRCA1 and 53BP1 tumour suppressor proteins. Unlike core components of the DNA repair machinery that interact with or enzymatically process the DNA, these proteins exert their influence indirectly, interacting with large regions of chromatin spanning single DSBs. However, the respective activities of these proteins and nature of chromatin changes that are brought about by their enrichment at DNA damage sites remain undefined.
Recent research has revealed that BRCA1 functions to antagonize 53BP1-dependent DSB repair activities during S-phase. Furthermore, an inability to counteract 53BP1 results in the chromosomal instability and tumour predisposition evident in cellular and mouse models of Brca1-deficiency, respectively. Our recent work has focused on understanding the opposing molecular roles of the BRCA1 and 53BP1 proteins in regulating DNA double-strand break repair pathway choice. We have also recently identified RIF1 as the major effector protein during 53BP1-dependent non-homologous end joining, a process crucial for humoral immunity that also drives genomic instability in cells lacking functional BRCA1.
Using transgenic mouse models and a combination of cell biology, biochemical, genomic and proteomic approaches, we are investigating the alterations that occur within DSB-associated chromatin as a result of the activities of these core proteins and other newly identified components of the DNA damage response. Moreover, we hope to elucidate how such changes determine DSB repair fate, to better understand why a breakdown in these processes can result in disease and cancer predisposition in humans.
There are no collaborations listed for this principal investigator.
2013. RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection. Mol Cell, 49 (5), pp. 858-871. Read abstract | Read more
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
2012. Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair. Mol Cell, 45 (4), pp. 505-516. Read abstract | Read more
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. Hide abstract
2012. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J Cell Sci, 125 (Pt 15), pp. 3529-3534. Read abstract | Read more
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
2009. A Supramodular FHA/BRCT-Repeat Architecture Mediates Nbs1 Adaptor Function in Response to DNA Damage Cell, 139 (1), pp. 100-111. | Read more
2008. DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev, 22 (20), pp. 2767-2772. Read abstract | Read more
A key cellular response to DNA double-strand breaks (DSBs) is 5'-to-3' DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLM--whose deficiency causes cancer predisposition and infertility in people--also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair. Hide abstract
2008. Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage. EMBO Rep, 9 (8), pp. 795-801. Read abstract | Read more
Mammalian cells respond to DNA double-strand breaks (DSBs) by recruiting DNA repair and cell-cycle checkpoint proteins to such sites. Central to these DNA damage response (DDR) events is the DNA damage mediator protein MDC1. MDC1 interacts with several DDR proteins, including the MRE11-RAD50-NBS1 (MRN) complex. Here, we show that MDC1 is phosphorylated on a cluster of conserved repeat motifs by casein kinase 2 (CK2). Moreover, we establish that this phosphorylation of MDC1 promotes direct, phosphorylation-dependent interactions with NBS1 in a manner that requires the closely apposed FHA and twin BRCT domains in the amino terminus of NBS1. Finally, we show that these CK2-targeted motifs in MDC1 are required to mediate NBS1 association with chromatin-flanking sites of unrepaired DSBs. These findings provide a molecular explanation for the MDC1-MRN interaction and yield insights into how MDC1 coordinates the focal assembly and activation of several DDR factors in response to DNA damage. Hide abstract
2007. Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase Science, 318 (5856), pp. 1637-1640. | Read more
2012. Playing the End Game: DNA Double-Strand Break Repair Pathway Choice Molecular Cell, 47 (4), pp. 497-510. Read abstract | Read more
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. © 2012 Elsevier Inc. Hide abstract
2003. XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus. Mol Biol Cell, 14 (9), pp. 3898-3910. Read abstract | Read more
The DNA damage/replication checkpoints act by sensing the presence of damaged DNA or stalled replication forks and initiate signaling pathways that arrest cell cycle progression. Here we report the cloning and characterization of Xenopus orthologues of the RFCand PCNA-related checkpoint proteins. XRad17 shares regions of homology with the five subunits of Replication factor C. XRad9, XRad1, and XHus1 (components of the 9-1-1 complex) all show homology to the DNA polymerase processivity factor PCNA. We demonstrate that these proteins associate with chromatin and are phosphorylated when replication is inhibited by aphidicolin. Phosphorylation of X9-1-1 is caffeine sensitive, but the chromatin association of XRad17 and the X9-1-1 complex after replication block is unaffected by caffeine. This suggests that the X9-1-1 complex can associate with chromatin independently of XAtm/XAtr activity. We further demonstrate that XRad17 is essential for the chromatin binding and checkpoint-dependent phosphorylation of X9-1-1 and for the activation of XChk1 when the replication checkpoint is induced by aphidicolin. XRad17 is not, however, required for the activation of XCds1 in response to dsDNA ends. Hide abstract
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