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      The RSF1 Histone-Remodelling Factor Facilitates DNA Double-Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins

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      PLoS Biology

      Public Library of Science

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          Abstract

          RSF1, a new player in the cellular responses to DNA double-strand breaks, sequentially recruits centromeric histone-like proteins and DNA repair proteins from the Fanconi anaemia pathway.

          Abstract

          ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM–RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair.

          Author Summary

          DNA carries all the information necessary for life; thus damage or loss of genetic material can result in cell death or cancer. The worst-case insult to genetic information is a DNA double-strand break, caused by agents either within the cell (e.g., by-products of respiration, errors of DNA replication) or from outside (e.g., ionizing radiation). Ataxia telangiectasia kinase (ATM) and the Fanconi anaemia proteins perform housekeeping roles in the cell, recognising aberrant DNA structures and promoting their repair. Mutations that affect these proteins are responsible for the eponymous genetic syndromes that are characterised by elevated mutation rate, increased cancer risk, developmental defects, and shortened life span. In this work we identify and characterise a novel link between these two central players in the DNA damage response. We show that the Remodelling and Spacing Factor 1 (RSF1) protein, which can reorganise the compaction of DNA to allow access for other proteins, requires ATM for its function after DNA damage. Specifically, RSF1 recruits two centromeric histone-like proteins that in turn promote mono-ubiquitination and recruitment to sites of damage of FANCD2 and FANCI—two proteins that belong to the Fanconi anaemia pathway. Absence of RSF1 results in defective repair of double-strand DNA breaks, prolonged arrest of the cell cycle, and cell death. Our study suggests that ATM-dependent regulation of the RSF chromatin-remodelling complex is necessary during double-strand break repair to recruit centromeric histones and then Fanconi anaemia proteins.

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          Most cited references 51

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          MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

          Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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            Andromeda: a peptide search engine integrated into the MaxQuant environment.

            A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spectra Andromeda is also accessible via a web server. We demonstrate the flexibility of the system by implementing the capability to identify cofragmented peptides, significantly improving the total number of identified peptides.
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              The DNA-damage response in human biology and disease.

              The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                May 2014
                6 May 2014
                : 12
                : 5
                Affiliations
                Genome Stability Laboratory, Centre for Chromosome Biology, School of Natural Science, National University of Ireland Galway, Ireland
                Dana-Farber Cancer Institute, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: NFL FP. Performed the experiments: FP. Analyzed the data: NFL FP. Wrote the paper: NFL FP.

                Article
                PBIOLOGY-D-13-05006
                10.1371/journal.pbio.1001856
                4011676
                24800743

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 17
                Funding
                This work was supported by a Science Foundation Ireland ( http://www.sfi.ie/) Principal Investigator award (07/IN1/B958) to NFL, a European Union DNA repair ( http://www.dna-repair.nl/) FP6 Integrated Project (contract 512113) to NFL, and a Health Research Board ( http://www.hrb.ie) Programme Award (PR001/2001) to NFL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteomics
                Cell Biology
                Molecular Cell Biology
                Genetics
                Radiobiology

                Life sciences

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