DNA damage signaling in response to double-strand breaks during mitosis

Dividing cells can sense DNA damage and initiate a primary response, but repair isn’t completed until the cells enter G1

PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1

Abstract: Loss of functional BRCA1 protein leads to defects in DNA double-strand break (DSB) repair by homologous recombination (HR) and renders cells hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors used to treat BRCA1/2-deficient cancers. However, upon chronic treatment of BRCA1-mutant cells with PARP inhibitors, resistant clones can arise via several mechanisms, including loss of 53BP1 or its downstream co-factors. Defects in the 53BP1 axis partially restore the ability of a BRCA1-deficient cell to form RAD51 filaments at resected DSBs in a PALB2- and BRCA2-dependent manner, and thereby repair DSBs by HR. Here we show that depleting 53BP1 in BRCA1-null cells restores PALB2 accrual at resected DSBs. Moreover, we demonstrate that PALB2 DSB recruitment in BRCA1/53BP1-deficient cells is mediated by an interaction between PALB2’s chromatin associated motif (ChAM) and the nucleosome acidic patch region, which in 53BP1-expressing cells is bound by 53BP1’s ubiquitin-directed recruitment (UDR) domain

Small Molecule Inhibition of UBE2T/FANCL-mediated Ubiquitylation in the Fanconi Anemia Pathway

The Fanconi anemia pathway orchestrates the repair of DNA inter-strand crosslinks and stalled replication forks. A key step in this pathway is UBE2T and FANCL dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, very few selective inhibitors of ubiquitin conjugation pathways are known to date. By using a high-throughput screen compatible assay, we have identified a small molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.M.J.C was funded through the Cambridge PhD Training Programme in Chemical Biology and Molecular Medicine. Y.G is funded by Cancer Research UK, C6/A18796 and a Wellcome Trust Investigator Award to S.P.J. 206388/Z/17/Z. Research in the Jackson lab is funded by CRUK (C6/A18796) and the Wellcome Trust (206388/Z/17/Z), with core infrastructure funding from the Wellcome Trust (203144) and CRUK (C6946/A24843). Work in the CRUK Manchester Institute Drug Discovery Unit was funded by CRUK (Grant numbers C480/A1141 and C309/A8274)

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

Replication stress induces 53BP1-containing OPT domains in G1 cells

53BP1-OPT domains, nuclear bodies that arise in G1 cells at sites of DNA damage induced by incomplete DNA replication, preferentially localize to chromosomal common fragile sites

CtIP tetramer assembly is required for DNA-end resection and repair.

Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.We thank M. Kilkenny for help with the collection of X-ray diffraction data, A. Sharff and P. Keller for help with X-ray data processing and J.D. Maman for assistance with SEC-MALS. This work was supported by a Wellcome Trust Senior Research Fellowship award in basic biomedical sciences (L.P.), an Isaac Newton Trust research grant (L.P. and O.R.D.) and a Cambridge Overseas Trust PhD studentship (M.D.S.). Research in the laboratory of S.P.J. is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. R.B. and J.C. are funded by Cancer Research UK programme grant C6/A11224. Y.G. and M.D. are funded by the European Research Council grant DDREAM.This is the accepted manuscript of a paper published in Nature Structural & Molecular Biology, 22, 150–157 (2015) doi: 10.1038/nsmb.293

Multiple roles of the histone acetyltransferase complex SAGA in RNA polymerase II -dependent transcription in yeast Saccharomyces cerevisiae

Transcription initiation in the eukaryotic cell is a complex process which requires multiple protein-DNA and protein-protein contacts to be established. In addition, the access of transcription factors to DNA is obscured by chromatin whose repressive effects can be overcome by chromatin modifying or remodeling enzymes. The incorporation of these enzymes into multisubunit complexes leads to the important questions what roles other components of the complexes might play and whether these complexes are changing in response to different stimuli. Both issues have been addressed in this study focused on the yeast SAGA complex. SAGA is a 1.8 MDa yeast protein complex composed of several distinct classes of transcription-related factors, including the adaptor/histone acetyltransferase Gcn5, Spt proteins, a subset of TafIIs and the ATM and DNA-PK-related Tra1 protein. Yeast SAGA satisfies the definition of a typical coactivator complex, because it can interact both with acidic activators and with at least one general transcription factor, TBP. Most of the information about SAGA functions was obtained in in vitro experiments. In this study, in vivo functions of SAGA have been investigated. First, the roles of different SAGA subunits at endogenous yeast promoters have been examined. Mutations that completely disrupt SAGA (deletions of SPT7or SPT20) strongly reduce transcriptional activation of a number of genes suggesting that the complex integrity is important for transcriptional regulation. HAT activity of Gcn5 is required for SAGA coactivator function, as well as for normal start site selection at HIS3 promoter. Surprisingly, mutations in Spt proteins involved in SAGA/TBP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcription in the uninduced state. Consistent with this, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro while SAGA lacking Spt3 or Spt8 is not inhibitory. Thus, different SAGA components have distinct roles in transcriptional regulation, both stimulatory and inhibitory. The second major finding of this study is that a distinct form of SAGA has been detected upon induction of a particular pathway in which its activity is required. This novel complex contains electrophoretically altered Spt7 subunit and lacks two proteins, Spt8 and Sin4, that can both function as transcriptional inhibitors. Thus, SAGA appears to function in the fine tuning of transcription at specific promoters and its subunit composition might be dynamic, changing under different physiological conditions. Structural changes in SAGA may represent a paradigm for the regulation of multisubunit chromatin-modifying complexes

Multiple roles of the histone acetyltransferase complex SAGA in RNA polymerase II -dependent transcription in yeast Saccharomyces cerevisiae

Transcription initiation in the eukaryotic cell is a complex process which requires multiple protein-DNA and protein-protein contacts to be established. In addition, the access of transcription factors to DNA is obscured by chromatin whose repressive effects can be overcome by chromatin modifying or remodeling enzymes. The incorporation of these enzymes into multisubunit complexes leads to the important questions what roles other components of the complexes might play and whether these complexes are changing in response to different stimuli. Both issues have been addressed in this study focused on the yeast SAGA complex. SAGA is a 1.8 MDa yeast protein complex composed of several distinct classes of transcription-related factors, including the adaptor/histone acetyltransferase Gcn5, Spt proteins, a subset of TafIIs and the ATM and DNA-PK-related Tra1 protein. Yeast SAGA satisfies the definition of a typical coactivator complex, because it can interact both with acidic activators and with at least one general transcription factor, TBP. Most of the information about SAGA functions was obtained in in vitro experiments. In this study, in vivo functions of SAGA have been investigated. First, the roles of different SAGA subunits at endogenous yeast promoters have been examined. Mutations that completely disrupt SAGA (deletions of SPT7or SPT20) strongly reduce transcriptional activation of a number of genes suggesting that the complex integrity is important for transcriptional regulation. HAT activity of Gcn5 is required for SAGA coactivator function, as well as for normal start site selection at HIS3 promoter. Surprisingly, mutations in Spt proteins involved in SAGA/TBP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcription in the uninduced state. Consistent with this, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro while SAGA lacking Spt3 or Spt8 is not inhibitory. Thus, different SAGA components have distinct roles in transcriptional regulation, both stimulatory and inhibitory. The second major finding of this study is that a distinct form of SAGA has been detected upon induction of a particular pathway in which its activity is required. This novel complex contains electrophoretically altered Spt7 subunit and lacks two proteins, Spt8 and Sin4, that can both function as transcriptional inhibitors. Thus, SAGA appears to function in the fine tuning of transcription at specific promoters and its subunit composition might be dynamic, changing under different physiological conditions. Structural changes in SAGA may represent a paradigm for the regulation of multisubunit chromatin-modifying complexes

Elongation by RNA polymerase II: the short and long of it.

Appreciable advances into the process of transcript elongation by RNA polymerase II (RNAP II) have identified this stage as a dynamic and highly regulated step of the transcription cycle. Here, we discuss the many factors that regulate the elongation stage of transcription. Our discussion includes the classical elongation factors that modulate the activity of RNAP II, and the more recently identified factors that facilitate elongation on chromatin templates. Additionally, we discuss the factors that associate with RNAP II, but do not modulate its catalytic activity. Elongation is highlighted as a central process that coordinates multiple stages in mRNA biogenesis and maturation