Begg, K.A.
Braun, H.
Ghaddar, N.
Wu, L.
Downs, J.A.
(2024). Defects in DNA damage responses in SWI/SNF mutant cells and their impact on immune responses. Dna repair,
Vol.133,
pp. 103609-103609.
full text
Schiavoni, F.
Zuazua-Villar, P.
Roumeliotis, T.I.
Benstead-Hume, G.
Pardo, M.
Pearl, F.M.
Choudhary, J.S.
Downs, J.A.
(2022). Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness. Nat commun,
Vol.13
(1),
p. 1731.
show abstract
full text
Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy..
Feng, H.
Lane, K.A.
Roumeliotis, T.I.
Jeggo, P.A.
Somaiah, N.
Choudhary, J.S.
Downs, J.A.
(2022). PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance. Genes dev,
Vol.36
(13-14),
pp. 790-806.
show abstract
full text
The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer..
Chabanon, R.M.
Morel, D.
Eychenne, T.
Colmet-Daage, L.
Bajrami, I.
Dorvault, N.
Garrido, M.
Meisenberg, C.
Lamb, A.
Ngo, C.
Hopkins, S.R.
Roumeliotis, T.I.
Jouny, S.
Hénon, C.
Kawai-Kawachi, A.
Astier, C.
Konde, A.
Del Nery, E.
Massard, C.
Pettitt, S.J.
Margueron, R.
Choudhary, J.S.
Almouzni, G.
Soria, J.-.
Deutsch, E.
Downs, J.A.
Lord, C.J.
Postel-Vinay, S.
(2021). PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer. Cancer res,
Vol.81
(11),
pp. 2888-2902.
show abstract
Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg..
Wilkins, A.
Fontana, E.
Nyamundanda, G.
Ragulan, C.
Patil, Y.
Mansfield, D.
Kingston, J.
Errington-Mais, F.
Bottomley, D.
von Loga, K.
Bye, H.
Carter, P.
Tinkler-Hundal, E.
Noshirwani, A.
Downs, J.
Dillon, M.
Demaria, S.
Sebag-Montefiore, D.
Harrington, K.
West, N.
Melcher, A.
Sadanandam, A.
(2021). Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer. J immunother cancer,
Vol.9
(3).
show abstract
full text
BACKGROUND: Rectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer. METHODS: We generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses. RESULTS: ΔTCD scores ranged from 12.4% to -47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders. CONCLUSION: This study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune "cold" towards an immunologically "hot" phenotype on treatment with radiotherapy..
Flaus, A.
Downs, J.A.
Owen-Hughes, T.
(2021). Histone isoforms and the oncohistone code. Curr opin genet dev,
Vol.67,
pp. 61-66.
show abstract
full text
Recent studies have highlighted the potential for missense mutations in histones to act as oncogenic drivers, leading to the term 'oncohistones'. While histone proteins are highly conserved, they are encoded by multigene families. There is heterogeneity among these genes at the level of the underlying sequence, the amino acid composition of the encoded histone isoform, and the expression levels. One question that arises, therefore, is whether all histone-encoding genes function equally as oncohistones. In this review, we consider this question and explore what this means in terms of the mechanisms by which oncohistones can exert their effects in chromatin..
Lindenburg, L.H.
Pantelejevs, T.
Gielen, F.
Zuazua-Villar, P.
Butz, M.
Rees, E.
Kaminski, C.F.
Downs, J.A.
Hyvönen, M.
Hollfelder, F.
(2021). Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats. Proc natl acad sci u s a,
Vol.118
(46).
show abstract
full text
Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation..
Anbalagan, S.
Ström, C.
Downs, J.A.
Jeggo, P.A.
McBay, D.
Wilkins, A.
Rothkamm, K.
Harrington, K.J.
Yarnold, J.R.
Somaiah, N.
(2021). TP53 modulates radiotherapy fraction size sensitivity in normal and malignant cells. Sci rep,
Vol.11
(1),
p. 7119.
show abstract
full text
Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential to fully exploit this therapeutic variable for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is dependent on the presence of wild-type (WT) p53 and intact non-homologous end-joining (NHEJ). Using single or split-doses of radiation in a range of normal and malignant cells, split-dose recovery was determined using colony-survival assays. Both normal and tumour cells with WT p53 demonstrated significant split-dose recovery, whereas Li-Fraumeni fibroblasts and tumour cells with defective G1/S checkpoint had a large S/G2 component and lost the sparing effect of smaller fractions. There was lack of split-dose recovery in NHEJ-deficient cells and DNA-PKcs inhibitor increased sensitivity to split-doses in glioma cells. Furthermore, siRNA knockdown of p53 in fibroblasts reduced split-dose recovery. In summary, cells defective in p53 are less sensitive to radiotherapy fraction size and lack of split-dose recovery in DNA ligase IV and DNA-PKcs mutant cells suggests the dependence of fraction size sensitivity on intact NHEJ..
Harrod, A.
Lane, K.A.
Downs, J.A.
(2020). The role of the SWI/SNF chromatin remodelling complex in the response to DNA double strand breaks. Dna repair (amst),
Vol.93,
pp. 102919-102919.
show abstract
full text
Mammalian cells possess multiple closely related SWI/SNF chromatin remodelling complexes. These complexes have been implicated in the cellular response to DNA double strand breaks (DSBs). Evidence suggests that SWI/SNF complexes contribute to successful repair via both the homologous recombination and non-homologous end joining pathways. In addition, repressing transcription near DSBs is dependent on SWI/SNF activity. Understanding these roles is important because SWI/SNF complexes are frequently dysregulated in cancer, and DNA DSB repair defects have the potential to be therapeutically exploited. In this graphical review, we summarise what is known about SWI/SNF contribution to DNA DSB responses in mammalian cells and provide an overview of the SWI/SNF-encoding gene alteration spectrum in human cancers..
Rother, M.B.
Pellegrino, S.
Smith, R.
Gatti, M.
Meisenberg, C.
Wiegant, W.W.
Luijsterburg, M.S.
Imhof, R.
Downs, J.A.
Vertegaal, A.C.
Huet, S.
Altmeyer, M.
van Attikum, H.
(2020). CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks. Nat commun,
Vol.11
(1),
p. 5775.
show abstract
full text
Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled 'chromatin breathing' upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails..
Benstead-Hume, G.
Chen, X.
Hopkins, S.R.
Lane, K.A.
Downs, J.A.
Pearl, F.M.
(2019). Predicting synthetic lethal interactions using conserved patterns in protein interaction networks. Plos comput biol,
Vol.15
(4),
p. e1006888.
show abstract
full text
In response to a need for improved treatments, a number of promising novel targeted cancer therapies are being developed that exploit human synthetic lethal interactions. This is facilitating personalised medicine strategies in cancers where specific tumour suppressors have become inactivated. Mainly due to the constraints of the experimental procedures, relatively few human synthetic lethal interactions have been identified. Here we describe SLant (Synthetic Lethal analysis via Network topology), a computational systems approach to predicting human synthetic lethal interactions that works by identifying and exploiting conserved patterns in protein interaction network topology both within and across species. SLant out-performs previous attempts to classify human SSL interactions and experimental validation of the models predictions suggests it may provide useful guidance for future SSL screenings and ultimately aid targeted cancer therapy development..
Benstead-Hume, G.
Wooller, S.K.
Downs, J.A.
Pearl, F.M.
(2019). Defining Signatures of Arm-Wise Copy Number Change and Their Associated Drivers in Kidney Cancers. Int j mol sci,
Vol.20
(22).
show abstract
full text
Using pan-cancer data from The Cancer Genome Atlas (TCGA), we investigated how patterns in copy number alterations in cancer cells vary both by tissue type and as a function of genetic alteration. We find that patterns in both chromosomal ploidy and individual arm copy number are dependent on tumour type. We highlight for example, the significant losses in chromosome arm 3p and the gain of ploidy in 5q in kidney clear cell renal cell carcinoma tissue samples. We find that specific gene mutations are associated with genome-wide copy number changes. Using signatures derived from non-negative factorisation, we also find gene mutations that are associated with particular patterns of ploidy change. Finally, utilising a set of machine learning classifiers, we successfully predicted the presence of mutated genes in a sample using arm-wise copy number patterns as features. This demonstrates that mutations in specific genes are correlated and may lead to specific patterns of ploidy loss and gain across chromosome arms. Using these same classifiers, we highlight which arms are most predictive of commonly mutated genes in kidney renal clear cell carcinoma (KIRC)..
Meisenberg, C.
Pinder, S.I.
Hopkins, S.R.
Wooller, S.K.
Benstead-Hume, G.
Pearl, F.M.
Jeggo, P.A.
Downs, J.A.
(2019). Repression of Transcription at DNA Breaks Requires Cohesin throughout Interphase and Prevents Genome Instability. Mol cell,
Vol.73
(2),
pp. 212-223.e7.
show abstract
full text
Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer..
Jeggo, P.A.
Downs, J.A.
Gasser, S.M.
(2017). Chromatin modifiers and remodellers in DNA repair and signalling. Philos trans r soc lond b biol sci,
Vol.372
(1731).
full text
Hopkins, S.R.
McGregor, G.A.
Murray, J.M.
Downs, J.A.
Savic, V.
(2016). Novel synthetic lethality screening method identifies TIP60-dependent radiation sensitivity in the absence of BAF180. Dna repair (amst),
Vol.46,
pp. 47-54.
show abstract
In recent years, research into synthetic lethality and how it can be exploited in cancer treatments has emerged as major focus in cancer research. However, the lack of a simple to use, sensitive and standardised assay to test for synthetic interactions has been slowing the efforts. Here we present a novel approach to synthetic lethality screening based on co-culturing two syngeneic cell lines containing individual fluorescent tags. By associating shRNAs for a target gene or control to individual fluorescence labels, we can easily follow individual cell fates upon siRNA treatment and high content imaging. We have demonstrated that the system can recapitulate the functional defects of the target gene depletion and is capable of discovering novel synthetic interactors and phenotypes. In a trial screen, we show that TIP60 exhibits synthetic lethality interaction with BAF180, and that in the absence of TIP60, there is an increase micronuclei dependent on the level of BAF180 loss, significantly above levels seen with BAF180 present. Moreover, the severity of the interactions correlates with proxy measurements of BAF180 knockdown efficacy, which may expand its usefulness to addressing synthetic interactions through titratable hypomorphic gene expression..
Myrianthopoulos, V.
Gaboriaud-Kolar, N.
Tallant, C.
Hall, M.-.
Grigoriou, S.
Brownlee, P.M.
Fedorov, O.
Rogers, C.
Heidenreich, D.
Wanior, M.
Drosos, N.
Mexia, N.
Savitsky, P.
Bagratuni, T.
Kastritis, E.
Terpos, E.
Filippakopoulos, P.
Müller, S.
Skaltsounis, A.-.
Downs, J.A.
Knapp, S.
Mikros, E.
(2016). Discovery and Optimization of a Selective Ligand for the Switch/Sucrose Nonfermenting-Related Bromodomains of Polybromo Protein-1 by the Use of Virtual Screening and Hydration Analysis. J med chem,
Vol.59
(19),
pp. 8787-8803.
show abstract
full text
Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein-ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5)..
Meisenberg, C.
Ashour, M.E.
El-Shafie, L.
Liao, C.
Hodgson, A.
Pilborough, A.
Khurram, S.A.
Downs, J.A.
Ward, S.E.
El-Khamisy, S.F.
(2016). Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan. Nucleic acids res,
.
show abstract
full text
The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance..
Alatwi, H.E.
Downs, J.A.
(2015). Removal of H2A Z by INO80 promotes homologous recombination. Embo rep,
Vol.16
(8),
pp. 986-994.
show abstract
full text
The mammalian INO80 remodelling complex facilitates homologous recombination (HR), but the mechanism by which it does this is unclear. Budding yeast INO80 can remove H2A.Z/H2B dimers from chromatin and replace them with H2A/H2B dimers. H2A.Z is actively incorporated at sites of damage in mammalian cells, raising the possibility that H2A.Z may need to be subsequently removed for resolution of repair. Here, we show that H2A.Z in human cells is indeed rapidly removed from chromatin flanking DNA damage by INO80. We also report that the histone chaperone ANP32E, which is implicated in removing H2AZ from chromatin, similarly promotes HR and appears to work on the same pathway as INO80 in these assays. Importantly, we demonstrate that the HR defect in cells depleted of INO80 or ANP32E can be rescued by H2A.Z co-depletion, suggesting that H2A.Z removal from chromatin is the primary function of INO80 and ANP32E in promoting homologous recombination..
Brownlee, P.M.
Meisenberg, C.
Downs, J.A.
(2015). The SWI/SNF chromatin remodelling complex: Its role in maintaining genome stability and preventing tumourigenesis. Dna repair (amst),
Vol.32,
pp. 127-133.
show abstract
Genes encoding subunits of the two SWI/SNF chromatin remodelling complexes (BAF and PBAF) are mutated in almost 20% of all human cancers. In addition to a role in regulating transcription, recent work from our laboratory and others identified roles for both complexes in DNA damage responses and the maintenance of sister chromatid cohesion, which may have profound impacts on genome stability and contribute to its role as a tumour suppressor. Here, we review some of the transcription-independent functions of the SWI/SNF chromatin remodelling complex and discuss these in light of their potential relevance to tumourigenesis..
Niimi, A.
Hopkins, S.R.
Downs, J.A.
Masutani, C.
(2015). The BAH domain of BAF180 is required for PCNA ubiquitination. Mutat res,
Vol.779,
pp. 16-23.
show abstract
Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination..
Schalbetter, S.A.
Mansoubi, S.
Chambers, A.L.
Downs, J.A.
Baxter, J.
(2015). Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability. Proc natl acad sci u s a,
Vol.112
(33),
pp. E4565-E4570.
show abstract
Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin..
Kakarougkas, A.
Downs, J.A.
Jeggo, P.A.
(2015). The PBAF chromatin remodeling complex represses transcription and promotes rapid repair at DNA double-strand breaks. Mol cell oncol,
Vol.2
(1),
p. e970072.
show abstract
Transcription in the vicinity of DNA double-strand breaks (DSBs) is suppressed via a process involving ataxia telangiectasia mutated protein (ATM) and H2AK119 ubiquitylation.(1) We discuss recent findings that components of the Polybromo and Brahma-related gene 1 (BRG1)-associated factor (PBAF) remodeling complex and the polycomb repressive complex (PRC1/2) are also required.(2) Failure to activate transcriptional suppression impedes a rapid DSB repair process..
Kakarougkas, A.
Ismail, A.
Chambers, A.L.
Riballo, E.
Herbert, A.D.
Künzel, J.
Löbrich, M.
Jeggo, P.A.
Downs, J.A.
(2014). Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Mol cell,
Vol.55
(5),
pp. 723-732.
show abstract
full text
Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity..
Pal, M.
Morgan, M.
Phelps, S.E.
Roe, S.M.
Parry-Morris, S.
Downs, J.A.
Polier, S.
Pearl, L.H.
Prodromou, C.
(2014). Structural basis for phosphorylation-dependent recruitment of Tel2 to Hsp90 by Pih1. Structure,
Vol.22
(6),
pp. 805-818.
show abstract
Client protein recruitment to the Hsp90 system depends on cochaperones that bind the client and Hsp90 simultaneously and facilitate their interaction. Hsp90 involvement in the assembly of snoRNPs, RNA polymerases, PI3-kinase-like kinases, and chromatin remodeling complexes depends on the TTT (Tel2-Tti1-Tti2), and R2TP complexes-consisting of the AAA-ATPases Rvb1 and Rvb2, Tah1 (Spagh/RPAP3 in metazoa), and Pih1 (Pih1D1 in humans)-that together provide the connection to Hsp90. The biochemistry underlying R2TP function is still poorly understood. Pih1 in particular, at the heart of the complex, has not been described at a structural level, nor have the multiple protein-protein interactions it mediates been characterized. Here we present a structural and biochemical analysis of Hsp90-Tah1-Pih1, Hsp90-Spagh, and Pih1D1-Tel2 complexes that reveal a domain in Pih1D1 specific for binding CK2 phosphorylation sites, and together define the structural basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex..
Jeggo, P.A.
Downs, J.A.
(2014). Roles of chromatin remodellers in DNA double strand break repair. Exp cell res,
Vol.329
(1),
pp. 69-77.
show abstract
Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways..
López-Perrote, A.
Alatwi, H.E.
Torreira, E.
Ismail, A.
Ayora, S.
Downs, J.A.
Llorca, O.
(2014). Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases. J biol chem,
Vol.289
(33),
pp. 22614-22629.
show abstract
Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination..
Brownlee, P.M.
Chambers, A.L.
Cloney, R.
Bianchi, A.
Downs, J.A.
(2014). BAF180 promotes cohesion and prevents genome instability and aneuploidy. Cell rep,
Vol.6
(6),
pp. 973-981.
show abstract
full text
BAF180, a subunit of the PBAF chromatin remodeling complex, is frequently mutated in cancer. Although PBAF regulates transcription, it remains unclear whether this is what drives tumorigenesis in cells lacking BAF180. Based on data from yeast, we hypothesized that BAF180 may prevent tumorigenesis by promoting cohesion. Here, we show BAF180 is required for centromeric cohesion in mouse and human cells. Mutations identified in tumor samples are unable to support this activity, and also compromise cohesion-dependent functions in yeast. We provide evidence of genome instability in line with loss of cohesion, and importantly, we find dynamic chromosome instability following DNA damage in cells lacking BAF180. These data demonstrate a function for BAF180 in promoting genome stability that is distinct from its well-characterized role in transcriptional regulation, uncovering a potent mechanism for its tumor-suppressor activity..
Chambers, A.L.
Pearl, L.H.
Oliver, A.W.
Downs, J.A.
(2013). The BAH domain of Rsc2 is a histone H3 binding domain. Nucleic acids res,
Vol.41
(19),
pp. 9168-9182.
show abstract
Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction..
Chambers, A.L.
Ormerod, G.
Durley, S.C.
Sing, T.L.
Brown, G.W.
Kent, N.A.
Downs, J.A.
(2012). The INO80 chromatin remodeling complex prevents polyploidy and maintains normal chromatin structure at centromeres. Genes dev,
Vol.26
(23),
pp. 2590-2603.
show abstract
The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability..
Brownlee, P.M.
Chambers, A.L.
Oliver, A.W.
Downs, J.A.
(2012). Cancer and the bromodomains of BAF180. Biochem soc trans,
Vol.40
(2),
pp. 364-369.
show abstract
Chromatin remodelling complexes alter the structure of chromatin and have central roles in all DNA-templated activities, including regulation of gene expression and DNA repair. Mutations in subunits of the PBAF (polybromo/Brg1-associated factor) or SWI/SNF-B remodelling complex, including BAF180, are frequently associated with cancer. There are six potential acetyl-lysine-binding BDs (bromodomains) in BAF180, which may function to target the PBAF complex to promoters or sites of DNA repair. In the present review, we discuss what is currently known about the BDs of BAF180 and their potential significance in cancer..
Niimi, A.
Chambers, A.L.
Downs, J.A.
Lehmann, A.R.
(2012). A role for chromatin remodellers in replication of damaged DNA. Nucleic acids res,
Vol.40
(15),
pp. 7393-7403.
show abstract
In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells..
Chambers, A.L.
Downs, J.A.
(2012). The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair. Prog mol biol transl sci,
Vol.110,
pp. 229-261.
show abstract
In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants..
Chambers, A.L.
Brownlee, P.M.
Durley, S.C.
Beacham, T.
Kent, N.A.
Downs, J.A.
(2012). The two different isoforms of the RSC chromatin remodeling complex play distinct roles in DNA damage responses. Plos one,
Vol.7
(2),
p. e32016.
show abstract
The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains..
Foster, E.R.
Downs, J.A.
(2009). Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation. J cell biol,
Vol.184
(5),
pp. 631-638.
show abstract
Covalent modifications of histone proteins have profound consequences on chromatin structure and function. Specific modification patterns constitute a code read by effector proteins. Studies from yeast found that H3 trimethylation at K4 and K79 is dependent on ubiquitylation of H2B K123, which is termed a "trans-tail pathway." In this study, we show that a strain unable to be ubiquitylated on H2B (K123R) is still proficient for H3 trimethylation at both K4 and K79, indicating that H3 methylation status is not solely dependent on H2B ubiquitylation. However, additional mutations in H2B result in loss of H3 methylation when combined with htb1-K123R. Consistent with this, we find that the original strain used to identify the trans-tail pathway has a genomic mutation that, when combined with H2B K123R, results in defective H3 methylation. Finally, we show that strains lacking the ubiquitin ligase Bre1 are defective for H3 methylation, suggesting that there is an additional Bre1 substrate that in combination with H2B K123 facilitates H3 methylation..
Titman, C.M.
Downs, J.A.
Oliver, S.G.
Carmichael, P.L.
Scott, A.D.
Griffin, J.L.
(2009). A metabolomic and multivariate statistical process to assess the effects of genotoxins in Saccharomyces cerevisiae. Mol biosyst,
Vol.5
(12),
pp. 1913-1924.
show abstract
There is an increased need to develop robust cellular model systems which could replace or reduce the need for animals in toxicological testing. Current in vitro strategies for genotoxicity testing suffer from a high irrelevant positive rate, requiring the need for the development of new in vitro tools. Saccharomyces cerevisiae is used widely to study DNA damage and repair, and a high-throughput green fluorescent protein based assay has been developed to detect genotoxic-induced DNA damage. In this study a combined high resolution (1)H NMR spectroscopy and gas chromatography mass spectrometry based metabolomic approach has been used to monitor and distinguish different genotoxic compounds from other types of toxic lesion using the multivariate classification tool partial least squares-discriminate analysis (PLS-DA). The metabolic profiles of extracts of yeast (W303alpha strain) readily distinguished the individual toxins from control cells across 22 different treatments. In addition, these metabolic profiles also demonstrated dose and time responses for selected compounds (methyl methane sulfonate and nocodazole). Finally, predictive models were built for distinguishing the genotoxic carcinogens from the control group according to the metabolic profile of the cell culture media..
Downs, J.A.
(2008). Histone H3 K56 acetylation, chromatin assembly, and the DNA damage checkpoint. Dna repair (amst),
Vol.7
(12),
pp. 2020-2024.
show abstract
The role of chromatin and its modulation during DNA repair has become increasingly understood in recent years. A number of histone modifications that contribute towards the cellular response to DNA damage have been identified, including the acetylation of histone H3 at lysine 56. H3 K56 acetylation occurs normally during S phase, but persists in the presence of DNA damage. In the absence of this modification, cellular survival following DNA damage is impaired. Two recent reports provide additional insights into how H3 K56 acetylation functions in DNA damage responses. In particular, this modification appears to be important for both normal replication-coupled nucleosome assembly as well as nucleosome assembly at sites of DNA damage following repair..
Bilsland, E.
Hult, M.
Bell, S.D.
Sunnerhagen, P.
Downs, J.A.
(2007). The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage. Dna repair (amst),
Vol.6
(10),
pp. 1471-1484.
show abstract
The ubiquitination status of proteins can control numerous aspects of protein function through targeted destruction or by altering protein-protein interactions, subcellular localization, or enzymatic activity. In addition to enzymes that mediate the conjugation of ubiquitin moieties to target proteins, there are enzymes that catalyze the removal of ubiquitin, termed ubiquitin proteases. One such ubiquitin protease, Ubp3, exists in a complex with a partner protein: Bre5. This complex has been implicated in a variety of cellular activities, and was recently identified in large-scale screens for genetic interactions with known components of the DNA damage response pathway. We found that this complex plays a role in the cellular response to the DNA damaging agent phleomycin and strains lacking the complex have a defect in non-homologous end joining. Although this complex is also important for telomeric silencing, maintenance of the cell wall, and global transcriptional regulation, we present evidence suggesting that the role of this complex in DNA damage responses is distinct from these other roles. First, we found that Ubp3/Bre5 functions antagonistically with Bul1 in DNA damage responses, but not in its other cellular functions. Additionally, we have generated mutants of Bre5 that are specifically defective in DNA damage responses..
Downs, J.A.
(2007). Chromatin structure and DNA double-strand break responses in cancer progression and therapy. Oncogene,
Vol.26
(56),
pp. 7765-7772.
show abstract
Defects in the detection and repair of DNA double-strand breaks (DSBs) have been causatively linked to tumourigenesis. Moreover, inhibition of DNA damage responses (DDR) can increase the efficacy of cancer therapies that rely on generation of damaged DNA. DDR must occur within the context of chromatin, and there have been significant advances in recent years in understanding how the modulation and manipulation of chromatin contribute to this activity. One particular covalent modification of a histone variant--the phosphorylation of H2AX--has been investigated in great detail and has been shown to have important roles in DNA DSB responses and in preventing tumourigenesis. These studies are reviewed here in the context of their relevance to cancer therapy and diagnostics. In addition, there is emerging evidence for contributions by proteins involved in mediating higher order structure to DNA DSB responses. The contributions of a subset of these proteins--linker histones and high-mobility group box (HMGB) proteins--to DDR and their potential significance in tumourigenesis are discussed..
Chambers, A.L.
Downs, J.A.
(2007). The contribution of the budding yeast histone H2A C-terminal tail to DNA-damage responses. Biochem soc trans,
Vol.35
(Pt 6),
pp. 1519-1524.
show abstract
The cellular response to DNA damage involves extensive interaction with and manipulation of chromatin. This includes the detection and repair of the DNA lesion, but there are also transcriptional responses to DNA damage, involving the up- or down-regulation of numerous genes. Therefore changes to chromatin structure, including covalent modification of histone proteins, are known to occur during DNA-damage responses. One of the most well characterized DNA-damage-responsive chromatin modification events is the phosphorylation of the SQ motif found in the C-terminal tail of histone H2A or the H2AX variant in higher eukaryotes. In the budding yeast, a number of additional residues in this region of histone H2A that contribute to the cellular response to DNA damage have been identified, providing an insight into the nature and complexity of the DNA-damage histone code..
Downs, J.A.
Nussenzweig, M.C.
Nussenzweig, A.
(2007). Chromatin dynamics and the preservation of genetic information. Nature,
Vol.447
(7147),
pp. 951-958.
show abstract
The integrity of the genome is frequently challenged by double-strand breaks in the DNA. Defects in the cellular response to double-strand breaks are a major cause of cancer and other age-related pathologies; therefore, much effort has been directed at understanding the enzymatic mechanisms involved in recognizing, signalling and repairing double-strand breaks. Recent work indicates that chromatin - the fibres into which DNA is packaged with a proteinaceous structural polymer - has an important role in initiating, propagating and terminating this cellular response to DNA damage..
Kent, N.A.
Chambers, A.L.
Downs, J.A.
(2007). Dual chromatin remodeling roles for RSC during DNA double strand break induction and repair at the yeast MAT locus. J biol chem,
Vol.282
(38),
pp. 27693-27701.
show abstract
DNA double strand breaks (DSBs) are potentially serious chromosomal lesions. However, cells sometimes deliberately cleave their own DNA to facilitate certain chromosomal processes, and there is much interest in how such self-inflicted breaks are effectively managed. Eukaryotic DSBs occur in the context of chromatin and the RSC chromatin-remodeling ATPase complex has been shown to promote DSB repair at the budding yeast MAT locus DSB, created by the HO endonuclease during mating type switching. We show that the role of RSC at MAT is highly specialized. The Rsc1p subunit of RSC directs nucleosome sliding immediately after DSB creation at both MAT and generally and is required for efficient DNA damage-induced histone H2A phosphorylation and strand resection during repair by homologous recombination. However, the Rsc2p and Rsc7p subunits are additionally required to set up a basal MAT locus structure. This RSC-dependent chromatin structure at MAT ensures accessibility to the HO endonuclease. The RSC complex therefore has chromatin remodeling roles both before and after DSB induction at MAT, promoting both DNA cleavage and subsequent repair..
Bilsland, E.
Downs, J.A.
(2005). Tails of histones in DNA double-strand break repair. Mutagenesis,
Vol.20
(3),
pp. 153-163.
show abstract
DNA double-strand breaks (DSBs) are, arguably, the most deleterious form of DNA damage. An increasing body of evidence points to the inaccurate or inefficient repair of DSBs as a key step in tumorigenesis. Therefore, it is of great importance to understand the processes by which DSBs are detected and repaired. Clearly, these events must take place in the context of chromatin in vivo, and recently, a great deal of progress has been made in understanding the dynamic and active role that histone proteins and chromatin modifying activities play in DNA DSB repair. Here, we briefly review some of the most common techniques in studying DNA DSB responses in vivo, and focus on the contributions of covalent modifications of core histone proteins to these DNA DSB responses..
Harvey, A.C.
Jackson, S.P.
Downs, J.A.
(2005). Saccharomyces cerevisiae histone H2A Ser122 facilitates DNA repair. Genetics,
Vol.170
(2),
pp. 543-553.
show abstract
DNA repair takes place in the context of chromatin. Recently, it has become apparent that proteins that make up and modulate chromatin structure are involved in the detection and repair of DNA lesions. We previously demonstrated that Ser129 in the carboxyl-terminal tail of yeast histone H2A is important for double-strand-break responses. By undertaking a systematic site-directed mutagenesis approach, we identified another histone H2A serine residue (Ser122) that is important for survival in the presence of DNA-damaging agents. We show that mutation of this residue does not affect DNA damage-dependent Rad53 phosphorylation or G(2)/M checkpoint responses. Interestingly, we find that yeast lacking H2A S122 are defective in their ability to sporulate. Finally, we demonstrate that H2A S122 provides a function distinct from that of H2A S129. These data demonstrate a role for H2A S122 in facilitating survival in the presence of DNA damage and suggest a potential role in mediating homologous recombination. The distinct roles of H2A S122 and S129 in mediating these responses suggest that chromatin components can provide specialized functions for distinct DNA repair and survival mechanisms and point toward the possibility of a complex DNA damage responsive histone code..
Foster, E.R.
Downs, J.A.
(2005). Histone H2A phosphorylation in DNA double-strand break repair. Febs j,
Vol.272
(13),
pp. 3231-3240.
show abstract
DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions..
Zaid, O.
Downs, J.A.
(2005). Histones as tumour suppressor genes. Cell mol life sci,
Vol.62
(15),
pp. 1653-1656.
show abstract
full text
Downs, J.A.
Côté, J.
(2005). Dynamics of chromatin during the repair of DNA double-strand breaks. Cell cycle,
Vol.4
(10),
pp. 1373-1376.
show abstract
Double strand breaks (DSBs) are arguably the most deleterious DNA lesion that a cell can sustain, and defects in the ability to detect and repair these breaks result in increased genomic instability and have been causatively linked to cancer. The repair of DNA DSBs must occur in the context of chromatin, and there is increasing evidence that the modulation of chromatin plays an integral role in the DNA DSB repair process. Here, we summarize a number of key findings, largely from studies performed in budding yeast, highlighting the role of chromatin in DNA DSB responses..
Wardleworth, B.N.
Downs, J.A.
(2005). Spotting new DNA damage-responsive chromatin-binding proteins. Biochem j,
Vol.388
(Pt 1),
pp. e1-e2.
show abstract
full text
In response to DNA damage, cells initiate multiple repair mechanisms that all contribute to the survival of both the cell and the organism. These responses are numerous and variable, and can include cell cycle arrest, transcriptional activation of DNA repair genes and relocalization of repair proteins to sites of DNA damage. If all else fails, in multicellular organisms the initiation of apoptosis is also a potential cellular response to DNA damage. Despite a wealth of information about these events, it is clear that we do not yet have a comprehensive picture of the cellular responses to DNA damage. In this issue of the Biochemical Journal, a proteomics approach was used by Lee et al. to identify proteins that bind to chromatin in a DNA damage-inducible manner. The proteins identified, nucleophosmin, hnRNP C1 (heterogeneous nuclear ribonucleoprotein C1) and hnRNP C2, were proteins that would not necessarily have been predicted to behave this way. These studies have the potential to be extended and contribute to our knowledge of the cellular response to DNA damage..
Downs, J.A.
Jackson, S.P.
(2004). A means to a DNA end: the many roles of Ku. Nat rev mol cell biol,
Vol.5
(5),
pp. 367-378.
Harvey, A.C.
Downs, J.A.
(2004). What functions do linker histones provide?. Mol microbiol,
Vol.53
(3),
pp. 771-775.
show abstract
Linker histones have long been known to facilitate chromatin condensation in vitro. For many years, this activity led to speculation that these proteins function as global repressors of transcription. However, work from numerous systems has demonstrated that this is not the case, and that linker histones appear to affect only the transcription of a small subset of genes, and, in fact, act to activate as well as repress transcription. Furthermore, there is evidence that suggests that linker histones are not required for mitotic condensation. In this review, we focus on studies of biological functions of linker histones primarily performed in unicellular eukaryotes. We highlight recent developments pointing towards linker histone involvement in DNA repair and ageing, which raise the possibility that the biological functions of linker histones are more varied than previously imagined..
Downs, J.A.
Allard, S.
Jobin-Robitaille, O.
Javaheri, A.
Auger, A.
Bouchard, N.
Kron, S.J.
Jackson, S.P.
Côté, J.
(2004). Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol cell,
Vol.16
(6),
pp. 979-990.
show abstract
Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair..
Downs, J.A.
Kosmidou, E.
Morgan, A.
Jackson, S.P.
(2003). Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone. Mol cell,
Vol.11
(6),
pp. 1685-1692.
show abstract
The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed..
Buratowski, R.M.
Downs, J.
Buratowski, S.
(2002). Interdependent interactions between TFIIB, TATA binding protein, and DNA. Mol cell biol,
Vol.22
(24),
pp. 8735-8743.
show abstract
full text
Temperature-sensitive mutants of TFIIB that are defective for essential interactions were isolated. One mutation (G204D) results in disruption of a protein-protein contact between TFIIB and TATA binding protein (TBP), while the other (K272I) disrupts an interaction between TFIIB and DNA. The TBP gene was mutagenized, and alleles that suppress the slow-growth phenotypes of the TFIIB mutants were isolated. TFIIB with the G204D mutation [TFIIB(G204D)] was suppressed by hydrophobic substitutions at lysine 239 of TBP. These changes led to increased affinity between TBP and TFIIB. TFIIB(K272I) was weakly suppressed by TBP mutants in which K239 was changed to hydrophobic residues. However, this mutant TFIIB was strongly suppressed by conservative substitutions in the DNA binding surface of TBP. Biochemical characterization showed that these TBP mutants had increased affinity for a TATA element. The TBPs with increased affinity could not suppress TFIIB(G204D), leading us to propose a two-step model for the interaction between TFIIB and the TBP-DNA complex..
Downs, J.A.
Lowndes, N.F.
Jackson, S.P.
(2000). A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature,
Vol.408
(6815),
pp. 1001-1004.
show abstract
Histone proteins associate with and compact eukaryotic nuclear DNA to form chromatin. The basic unit of chromatin is the nucleosome, which is made up of 146 base pairs of DNA wrapped around two of each of four core histones, H2A, H2B, H3 and H4. Chromatin structure and its regulation are important in transcription and DNA replication. We therefore thought that DNA-damage signalling and repair components might also modulate chromatin structure. Here we have characterized a conserved motif in the carboxy terminus of the core histone H2A from Saccharomyces cerevisiae that contains a consensus phosphorylation site for phosphatidylinositol-3-OH kinase related kinases (PIKKs). This motif is important for survival in the presence of agents that generate DNA double-strand breaks, and the phosphorylation of this motif in response to DNA damage is dependent on the PIKK family member Mec1. The motif is not necessary for Mec1-dependent cell-cycle or transcriptional responses to DNA damage, but is required for efficient DNA double-strand break repair by non-homologous end joining. In addition, the motif has a role in determining higher order chromatin structure. Thus, phosphorylation of a core histone in response to DNA damage may cause an alteration of chromatin structure that facilitates DNA repair..
Downs, J.A.
Jackson, S.P.
(1999). Involvement of DNA end-binding protein Ku in Ty element retrotransposition. Mol cell biol,
Vol.19
(9),
pp. 6260-6268.
show abstract
Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems..