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Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.

Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called ‘fast-kinetic’ canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either ‘slow-kinetic’ cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself.

Abscission, the final stage of cytokinesis, occurs when the cytoplasmic canal connecting two emerging daughter cells is severed either side of a large proteinaceous structure, the midbody. Here, we expand the functions of ATR to include a cell-cycle-specific role in abscission, which is required for genome stability. All previously characterized roles for ATR depend upon its recruitment to replication protein A (RPA)-coated single-stranded DNA (ssDNA). However, we establish that in each cell cycle ATR, as well as ATRIP, localize to the midbody specifically during late cytokinesis and independently of RPA or detectable ssDNA. Rather, midbody localization and ATR-dependent regulation of abscission requires the known abscission regulator-charged multivesicular body protein 4C (CHMP4C). Intriguingly, this regulation is also dependent upon the CDC7 kinase and the known ATR activator ETAA1. We propose that in addition to its known RPA-ssDNA-dependent functions, ATR has further functions in preventing premature abscission.

Ataxia Telangiectasia and Rad3 related (ATR) is one of the main regulators of the DNA damage response. It coordinates cell cycle checkpoint activation, replication fork stability, restart and origin firing to maintain genome integrity. Mutations of the ATR gene have been reported in Seckel patients, who suffer from a rare genetic disease characterized by severe microcephaly and growth retardation. Here, we report the case of a Seckel patient with compound heterozygous mutations in ATR. One allele has an intronic mutation affecting splicing of neighboring exons, the other an exonic missense mutation, producing the variant p.Lys1665Asn, of unknown pathogenicity. We have modeled this novel missense mutation, as well as a previously described missense mutation p.Met1159Ile, and assessed their effect on ATR function. Interestingly, our data indicate that both missense mutations have no direct effect on protein function, but rather result in defective ATR splicing. These results emphasize the importance of splicing mutations in Seckel Syndrome.

// Murilo T.D. Bueno 1, 2, 3, * , Marta Baldascini 4, * , Stephane Richard 1, 2, 3 and Noel F. Lowndes 4 1 Segal Cancer Center, Sir Mortimer B Davis Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada 2 Department of Medicine, McGill University, Montreal, Quebec, Canada 3 Department of Oncology, McGill University, Montreal, Quebec, Canada 4 Genome Stability Laboratory, Centre for Chromosome Biology, National University of Ireland, Galway, Ireland * These authors have contributed equally to this work Correspondence to: Stephane Richard, email: stephane.richard@mcgill.ca Noel F. Lowndes, email: noel.lowndes@nuigalway.ie Keywords: DNA double strand break repair; genome stability; KDM2A; 53BP1; ubiquitin Received: November 26, 2017 Accepted: February 27, 2018 Published: March 23, 2018 ABSTRACT Lysine demethylase 2A (KDM2A) functions in transcription as a demethylase of lysine 36 on histone H3. Herein, we characterise a role for KDM2A in the DNA damage response in which KDM2A stimulates conjugation of ubiquitin to 53BP1. Impaired KDM2A-mediated ubiquitination negatively affects the recruitment of 53BP1 to DSBs. Notably, we show that KDM2A itself is recruited to DSBs in a process that depends on its demethylase activity and zinc finger domain. Moreover, we show that KDM2A plays an important role in ensuring genomic stability upon DNA damage. Depletion of KDM2A or disruption of its zinc finger domain results in the accumulation of micronuclei following ionizing radiation (IR) treatment. In addition, IR-treated cells depleted of KDM2A display premature exit from the G2/M checkpoint. Interestingly, loss of the zinc finger domain also resulted in 53BP1 focal distribution in condensed mitotic chromosomes. Overall, our data indicates that KDM2A plays an important role in modulating the recruitment of 53BP1 to DNA breaks and is crucial for the preservation of genome integrity following DNA damage.

Summary: 53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.

Human blood monocytes are subclassified as classical, intermediate and nonclassical. In this study, it was shown that conventionally defined human intermediate monocytes can be divided into two distinct subpopulations with mid- and high-level surface expression of HLA-DR (referred to as DRmid and DRhi intermediate monocytes). These IM subpopulations were phenotypically and functionally characterized in healthy adult blood by flow cytometry, migration assays and lipoprotein uptake assays. Their absolute numbers and proportions were then compared in blood samples from obese and nonobese adults. DRmid and DRhi intermediate monocytes differentially expressed several proteins including CD62L, CD11a, CX3CR1 and CCR2. Overall, the DRmid intermediate monocytes surface profile more closely resembled that of classical monocytes while DRhi intermediate monocytes were more similar to nonclassical. However, in contrast to classical monocytes, DRmid intermediate monocytes migrated weakly to CCL2, had reduced intracellular calcium flux following CCR2 ligation and favored adherence to TNF alpha-activated endothelium over transmigration. In lipid uptake assays, DRmid intermediate monocytes demonstrated greater internalization of oxidized and acetylated low-density lipoprotein than DRhi intermediate monocytes. In obese compared to nonobese adults, proportions and absolute numbers of DRmid, but not DRhi intermediate monocytes, were increased in blood. The results are consistent with phenotypic and functional heterogeneity within the intermediate monocytes subset that may be of specific relevance to lipoprotein scavenging and metabolic health. The authors acknowledge the kind contribution of time and samples by study participants, as well as the assistance of the nursing and medical staff at the Centre for Diabetes, Endocrinology and Metabolism (CDEM) at Galway University Hospital. We also acknowledge assistance provided by Dr Paula O'Shea, Consultant Biochemist and staff at the Department of Biochemistry, Galway University Hospital. EPC, SN and SMS were supported by the Molecular Medicine Ireland Clinical and Translational Research Scholars Programme which was funded by the Irish Government's Programme for Research in Third Level Institutions, Cycle 5 and by NUI Galway. MCD was supported by the Health Services Executive (HSE) of Ireland, Medical Education, Training & Research (METR) Section and the Health Research Board (HRB) of Ireland under the National Specialist Registrar/Senior Registrar Academic Fellowship Program (NSAFP). JKE was partly‐funded by grants from the Irish Research Council for Science, Engineering and Technology (IRCSET EMPOWER fellowship) and the Health Research Board (grant number RP2006182N). Funding support was also received from Science Foundation Ireland [Regenerative Medicine Institute Strategic Research Cluster, grant number 09/SRC‐B1794; CÚRAM Research Centre, grant number 13/RC/2073 (MDG, TO'B, RC) and Principal Investigators grant numbers 07/IN1/B958 and 13/IA/1954 (NFL)], from the European Commission [Horizon 2020 Collaborative Health Project NEPHSTROM (Novel Stromal Cell Therapy for Diabetic Kidney Disease; grant 634086 (MDG, TO'B)] and from the European Regional Development Fund (all authors). All flow cytometry and cell sorting experiments were performed in the NUI Galway Flow Cytometry Core Facility which is supported by funds from NUI Galway, Science Foundation Ireland, the Irish Government's Programme for Research in Third Level Institutions, Cycle 5 and the European Regional Development Fund. peer-reviewed

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc(-)) DT40 cells arrest in G(2) phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc(-) cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G(2) checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G(2) arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G(2) phase.

In Saccharomyces cerevisiae, G1/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G1 phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G1/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region overrides the repression of MBF- and SBF-regulated genes by Nrm1 and Whi5. Finally, we show that the GTB motif is the core of a functional module that is necessary and sufficient for targeting of the transcription factors by their cognate repressors.

Thymic epithelial cells (TECs) are the main components of the thymic stroma that support and control T-cell development. Preparative regimens using DNA-damaging agents, such as total body irradiation and/or chemotherapeutic drugs, that are necessary prior to bone marrow transplantation (BMT) have profound deleterious effects on the hematopoietic system, including the thymic stroma, which may be one of the main causes for the prolonged periods of T-cell deficiency and the inefficient T cell reconstitution that are common following BMT. The DNA damage response (DDR) is a complex signaling network that allows cells to respond to all sorts of genotoxic insults. Hypoxia is known to modulate the DDR and play a role affecting the survival capacity of different cell types. In this study, we have characterized in detail the DDR of cortical and medullary TEC lines and their response to ionizing radiation, as well as the effects of hypoxia on their DDR. Although both mTECs and cTECs display relatively high radio-resistance, mTEC cells have an increased survival capacity to ionizing radiation (IR)-induced DNA damage, and hypoxia specifically decreases the radio-resistance of mTECs by upregulating the expression of the pro-apoptotic factor Bim. Analysis of the expression of TEC functional factors by primary mouse TECs showed a marked decrease of highly important genes for TEC function and confirmed cTECs as the most affected cell type by IR. These findings have important implications for improving the outcomes of BMT and promoting successful T cell reconstitution.

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae[1,2]. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologies with Rad24 [4], might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.

Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice.

Mesenchymal stromal cells (MSCs) are multi-potent adult stem cells located in various tissues, including the bone marrow. MSCs are key components of the haematopoietic stem cell (HSC) niche within the bone marrow where they function to maintain haematopoietic homoeostasis by regulating HSC self-renewal and function. Bone marrow exposure to ionising radiation causes rapid depletion of radio-sensitive HSCs and their progenitors, leading to haematopoietic failure. However, host-/patient-derived MSCs can survive radiation doses lethal to the haematopoietic system. The mechanisms underlying MSC radio-resistance are currently under intense investigation. Here, we review the current knowledge of MSC radio-biology. The DNA damage response (DDR) represents an orchestrated network of signalling pathways that enable cells to respond to genotoxic damage. We discuss in detail the emerging importance of the DDR in mediating MSC radio-resistance and examine the DDR of MSCs in the context of other stem cell types. Finally, we examine future advances in understanding MSC radio-resistance and discuss the potential impact of the radio-resistance of these stem cells for the clinic.

ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of DNA repair which permits replication to be restarted. However, the interplay between ATR and the FA pathway has been unclear. In this study, we present evidence that their action is directly linked, gaining insights into this relationship in a DT40 mutant cell line that is conditionally deficient in the critical ATR-binding partner protein ATRIP. Using this system, we showed that ATRIP was crucial for DNA damage–induced FANCD2 monoubiquitination and FANCI phosphorylation. ATR kinase phosphorylated recombinant FANCI protein in vitro, which was facilitated by the presence of FANCD2. Mechanistic investigations revealed that the RPA region but not the TopBP1 region of ATRIP was required for FANCD2 monoubiquitination, whereas Chk1 phosphorylation relied upon both domains. Together, our findings identify ATR as the kinase responsible for activating the FA pathway of DNA repair. Cancer Res; 72(5); 1149–56. ©2012 AACR.

The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1–14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017–1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G1 cells, declines throughout the S phase, increases again during late S/early G2, and persists through late G2 and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression. (SFI) Principal Investigator award (07/IN1/B958) and a Health Research Board (HRB) Project Grant (RP/2005/265) (to N. F. L.). Deposited by bulk import

2310 Cell Cycle 2007; Vol. 6 Issue 18DNA damage poses a constant threat to cell survival. In order to cope with the daily onslaught of genotoxic insults, cells have developed surveillance mechanisms that monitor DNA integrity, and in the presence of DNA damage activate checkpoint and repair pathways.

An integrated cellular response to DNA damage is essential for the maintenance of genome integrity. Recently, post-translational modifications to histone proteins have been implicated in DNA damage responses involving the Rad9 family of checkpoint proteins. In budding yeast, methylation of histone H3 on lysine 79 (H3-K79me) has been shown to be required for efficient checkpoint signalling and Rad9 localization on chromatin. Here, we have used a rad9 Tudor mutant allele and cells mutated for Dot1, the H3-K79 methylase, to analyse the epistatic relationship between RAD9 and DOT1 genes regarding the DNA damage resistance and checkpoint activation pathways. Our results show that RAD9 is epistatic to DOT1 and suggest that it acts downstream of the Dot1 methylase in the damage resistance and checkpoint response. We have also found that the Tudor domain of Rad9 is necessary for in vitro binding to H3-K79me as well as Rad9 focal accumulation in response to DNA damage in vivo. In summary, our study demonstrates that the interaction between Rad9, via its Tudor domain, and methylated H3-K79 is required at two different steps of the DNA damage response, an early step corresponding to checkpoint activation, and a late step corresponding to DNA repair. The study further shows that the function of this interaction is cell cycle-regulated; the role in checkpoint activation is restricted to the G1 phase and its role in DNA repair is restricted to G2. Copyright © 2007 John Wiley & Sons, Ltd.

A comprehensive meeting on biological responses to DNA damage organized by Alan Lehmann, Deborah Barnes, Wouter Ferro, Robert Fuchs, Jan Hoeijmakers, Roland Kanaar, Leon Mullenders and Bert van Zeeland, was convened at Noordwijkerhout, The Netherlands, from April 2 to 7, 2006. This article summarizes information presented by speakers at the seven plenary sessions. Poster sessions with organized discussions constituted a fundamental aspect of the meeting—as did a marvelous evening of musical entertainment by a talented group of conferees.

In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.

How phosphorylated histone H2AX, known as gamma-H2AX, functions in the cellular response to DNA double-strand breaks is the subject of intensive investigation. Recent research in yeast and mammalian cells shows that gamma-H2AX facilitates post-replicational DNA repair by recruiting cohesin, a protein complex that holds sister chromatids together.

Eukaryotic cells have evolved highly sophisticated cellular responses to DNA double strand breaks (DSBs) that increase the likelihood of survival. However, cells can also respond to DSBs by undergoing programmed cell death. The mechanisms underlying the cellular decision on whether to repair and survive or to die are not well understood but may be related to the efficiency of repair or the extent of the damage. Presumably, a few easily reparable DSBs will not result in cell death in most cell types. However, abundant complex DSBs will present a severe challenge to the repair machineries with repeated attempts at repair likely to result in genome instability. For multicellular eukaryotes at least, struggling to complete repair is folly, whereas removal of severely damaged cells is a more sensible strategy. Here we discuss recent evidence linking DSBs to a highly regulated form of cell death termed, apoptosis. In particular, we focus on the roles of the tumour suppressor, p53 and a recently discovered role for an isotype of the linker histone H1. We present a hypothesis that the elevated levels of ssDNA produced during ongoing attempts at DSB repair may be involved in the switch from repair to apoptosis.

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. The Rad9 protein functions in the DNA damage checkpoint pathway in Saccharomyces cerevisiae and is essential for the Mec1-dependent activation of the effector kinase Rad53. We recently described the purification of two soluble distinct Rad9 complexes. The large 850 kDa complex consists of hypophosphorylated Rad9 and the chaperone proteins Ssa1/2. This complex is found both in undamaged cells as well as in cells treated with DNA damaging agents. The smaller 560 kDa complex contains hyperphosphorylated Rad9, Ssa1/2 and, in addition, Rad53. This complex forms only in cells with compromised DNA integrity. Once bound to the smaller complex, Rad53 can be activated by in trans autophosphorylation. Here, we propose a model in which the large Rad9 complex is remodelled after a genomic insult by chaperone activity to a smaller Rad53 activating complex.

Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3) K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3) K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.

Eukaryotic cells have evolved surveillance mechanisms, known as DNA-damage checkpoints, that sense and respond to genome damage. DNA-damage checkpoint pathways ensure co-ordinated cellular responses to DNA damage, including cell cycle delays and activation of repair mechanisms. RAD9, from Saccharomyces cerevisiae, was the first damage checkpoint gene to be identified, although its biochemical function remained unknown until recently. This review examines briefly work that provides significant insight into how Rad9 activates the checkpoint signalling kinase Rad53.

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.

Mesenchymal stromal cells (MSCs) are radioresistant bone marrow progenitors that support hematopoiesis and its reconstitution following total body irradiation. MSCs reside in hypoxic niches within the bone marrow and tumor microenvironments. The DNA damage response (DDR) represents a network of signaling pathways that enable cells to activate biological responses to DNA damaging agents. Hypoxia-mediated alterations in the DDR contribute to the increased radioresistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radioresistance. However, the effects of hypoxia on MSC radioresistance are currently unknown. In this report, hypoxia was found to (a) increase MSC proliferation rate and colony size; (b) increase long-term survival post-irradiation (IR), and (c) improve MSC recovery from IR-induced cell cycle arrest. DNA double-strand break (DSB) repair in MSCs was upregulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA DSBs. In addition, HIF-1α was found to contribute to this enhanced DSB repair by regulating (a) the expression of DNA ligase IV and DNA-PKcs and (b) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radioresistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis. Stem Cells 2014;32:2188–2200

The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells. DOI: http://dx.doi.org/10.7554/eLife.03110.001, eLife digest Mutation can be harmful because changes to genes can disrupt vital processes or even cause diseases such as cancer. However, some genetic mutations can also be beneficial. Cells of the immune system, for example, need to create antibodies that attack a huge diversity of invading microbes. To do this, immune cells introduce changes into their genes to increase the diversity of the proteins that make up the antibodies. An enzyme called AID is thought to play a crucial role in increasing the diversity of our antibodies by changing specific letters of the genetic code. Now, Buerstedde et al. have shown that the AID enzyme can also cause sections of DNA to be deleted from the genome. Buerstedde et al. constructed pieces of DNA that include, in order, a gene that makes cells glow red, a gene that makes cells resistant to an antibiotic, and a gene that could make cells glow green. However, the very start of the ‘green’ gene was missing, which meant that it was switched off. Stretches of DNA were repeated in front of the ‘red’ and ‘green’ genes in some of the ‘constructs’. After inserting this DNA into cells from chickens or mice, most cells glowed red, but some started to glow green instead. Green cells were killed by the antibiotic; and were only seen when cells carried the constructs with the repeating DNA. Cells that lacked the AID enzyme only glowed red, regardless of which DNA construct they carried. Buerstedde et al. showed that AID causes the DNA constructs to align and re-arrange at the repeated sequences. As such, when the cells divide and their DNA is separated and packaged into newly formed cells, the DNA between the repeating sequences can be deleted. Thus, cells started to glow green because the ‘on’ switch at the start of the red gene ended up at the start of the green gene when the region in between was deleted. This also explains why green cells always died when exposed to the antibiotic, because this deletion removed the resistance gene too. Buerstedde et al. suggest that when a cell attempts to correct the errors caused by the AID enzyme changing the letters in the DNA, it actually can trigger the exchange and deletion of repeated sequences. Future work is now needed to understand how this new role for the AID enzyme is regulated, and whether this role beneficial or harmful to the immune cells. DOI: http://dx.doi.org/10.7554/eLife.03110.002

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., 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.

Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts. The larger (greater than or equal to 850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53. This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53. However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed. We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53. This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53.

In this study, the effect of a prior UV irradiation on the removal of cyclobutane pyrimidine dimers (CPDs) from the transcribed strand (TS) and non-transcribed strand (NTS) of the MFA2 gene in haploid Saccharomyces cerevisiae (S. cerevisiae) cells was investigated. In NER competent cells, the pre-irradiation with a dose of 20 J/m2 enhances the removal of CPDs induced by a second UV dose of 100 J/m2 in the TS and the NTS of MFA2 gene except for the CPDs in the region +258 to +298 in the NTS, where the enhanced repair was absent. No inducible repair was observed in rad9, rad24, rad16 and rad26 cells, indicating two checkpoint genes RAD9 and RAD24, the global repair gene RAD16 and the transcription coupled repair gene RAD26 are essential for inducible NER.

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 histones1, H2A, H2B, H3 and H4. Chromatin structure and its regulation are important in transcription and DNA replication2,3,4. 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.

Also known as… The Mre11 complex or MRX in yeast.What is MRN? A complex of three proteins — Mre11, Rad50 and Nbs1 (also known as Nibrin or p95). It is essential for the viability of vertebrate, but not yeast, cells. The MRN complex is engaged in DNA metabolic events involving DNA double-strand ends. Orthologues of human Rad50 and Mre11 have been identified in all taxonomic kingdoms whereas Nbs1 seems to be unique to eukaryotic cells as no orthologues have been identified in prokaryotes or archaebacteria. The well-characterized yeast homologue of MRN, MRX, contains the Mre11, Rad50 and Xrs2 proteins, the later showing weak homology to human Nbs1.Why is it essential for vertebrate cell viability? The genetic material of all eukaryotic cells is constantly exposed to both endogenous and exogenous DNA-damaging agents. Even a single double-stand break (DSB) can be lethal. Left unrepaired, DSBs can lead to chromosome instability, rearrangements, gene mutations and cancer. It is therefore extremely important for the cell to be able to sense the break, signal this damage and effect the appropriate biological responses as soon as possible. The MRN complex functions in both sensing and signaling of DSBs. It also has roles in both major DSB repair pathways — homologous recombination (HR) and non-homologous end joining (NHEJ). The MRN complex is also required for cell-cycle checkpoint signaling after DSB in all phases of the cell cycle. Additionally, it plays an important role in processing DNA structures that arise during normal S phase, is involved in preventing DNA re-replication and is essential for telomere maintenance.How do Mre11, Rad50 and Nbs1 contribute to MRN function? The three members of the complex have distinct roles within the intact MRN complex. Mre11 interacts with both Rad50 and Nbs1, which do not directly contact each other (Figure 1Figure 1). Rad50 has two globular domains linked by a long coiled-coil region forming extended arms. At the end of each arm a hook domain allows Rad50 molecules to dimerise and tether DNA ends together (Figure 1Figure 1). Mre11 is responsible for DNA binding and also has both exo- and endonuclease activities, which have been characterized in vitro, and an ability to unwind DNA locally. Finally, Nbs1, which has no known enzymatic activities, is responsible for the rapid re-localization of the complex into large focal structures, as well as for most of the interactions with other DSB-signaling and DNA-repair proteins. Its binding partners include ATM, γH2AX and MDC1. The carboxy-terminal region of Nbs1 has also been reported to regulate irradiation-induced apoptosis (Figure 1Figure 1).Figure 1Domain structure of the MRN components.Domains responsible for interactions within the complex are shown in yellow. CXXC hook, zinc hook; FHA, Forkhead associated domain; BRCT, BRCA1 carboxyl terminus domain. Note that the PAR domain of Mre11 localized between the two DNA binding motifs is not shown.View Large Image | View Hi-Res Image | Download PowerPoint SlideIs there a connection between MRN and cancer? Yes. Mre11, Rad50 and Nbs1 are known tumor suppressors. Loss of function of any of these proteins results in genome instability, the principal feature of cancer cells. Defective MRN function has been linked to many types of cancer, including breast, ovarian, colorectal, gastric and prostate cancers, as well as leukemia and melanoma. Hypomorphic mutations in any of the human genes for MRN result in cancer predisposing genome-instability syndromes: mutations in the Mre11 and Nbs1 genes cause ataxia telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Mutation of RAD50 has been described recently but so far there is no syndrome associated with it. The symptoms of MRN syndromes are largely overlapping and include immunodeficiency and mental deficiency.How does MRN respond to DNA damage? Immediately after the DSB induction, MRN re-localizes to the sites of damage (Figure 2Figure 2A). Initial recruitment is probably via its end-binding activity but, subsequently, excess MRN is recruited to the vicinity of DNA damage in large focal structures centered around DSBs. Focal accumulation is regulated by interaction with γH2AX, the DNA-damage-specific phosphoform of histone H2AX. During initial recruitment MRN is thought to bind and secure the DNA ends together via zinc hooks at the ends of the long flexible Rad50 arms, with the Mre11 molecules binding to DSBs (Figure 2Figure 2B). Upon DSB binding the Rad50 arms undergo structural change, becoming rigid and parallel and bridging both DNA ends using the zinc hook (Figure 2Figure 2B,D). Secured this way, the initial steps of DSB processing can take place. In addition, a signaling cascade is activated with the initial step being activation of the ATM kinase (Figure 2Figure 2D). Inactive ATM dimers are believed to be recruited to Nbs1 at DSBs, resulting in autophosphorylation and dissociation as active ATM monomers. The ATM kinase then phosphorylates many DNA-damage response proteins, including Nbs1 itself, resulting in the induction of the DNA-damage signaling cascade. Many DNA-damage response proteins are also recruited to the sites of damage and contribute to DSB processing and repair (Figure 2Figure 2C). ATM-dependent signaling contributes to efficient DSB repair, transcription and apoptosis, as well as regulating transient cell-cycle arrest, which is believed to allow sufficient time for DNA repair before the key cell-cycle transition.Figure 2A model presenting the initial steps of the DNA-damage response.(A) DSB induction. The MRN complex binds to DSBs and tethers DNA ends. PARP1 attaches ADP-ribose units to chromatin-bound proteins. Note that it is possible that DNA is secured by more than one copy of the complex at each DNA end. (B) The local concentration of MRN complex is increased by the interaction of Nbs1 with γH2AX and Mre11 binding to ADP-ribose units on chromatin-bound proteins. Note that the structure of the complex bound to DNA ends (rigid arms) differs from the unbound form (more flexible arms). At the same time, active ATM monomers phosphorylate downstream targets including Nbs1 and the signaling cascade is activated. (C) Repair proteins (including Brca1 and CtIP) are recruited to the site of damage. DNA ends are resected and the repair process begins. (D) Model illustrating structural changes in the MRN complex upon DNA binding and the role of MRN in the activation of the ATM kinase. The structure of chromatin-bound MRN complex remains unclear. Note that the MRN complex is composed of two Mre11 and Rad50 molecules and a single Nbs1 molecule.View Large Image | View Hi-Res Image | Download PowerPoint SlideAny surprising discoveries? The above model has recently been complicated still further by discoveries implicating poly(ADP-ribose) polymerase 1 (PARP1) in the rapid re-localisation of Mre11 and Nbs1 into focal structures at DNA damage sites (Figure 2Figure 2A–C). PARP1, which has not been directly linked to checkpoint activation, is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. Haince et al. suggest that Mre11 can directly bind to these ADP-ribose units via a short poly ADP-ribose (PAR) binding motif localized between two DNA-binding regions, contrasting with earlier models in which Nbs1, but not Mre11, is needed for localization of the MRN complex to the sites of damage. It is, however, possible that both proteins are needed for rapid, efficient accumulation of the complex at the sites of DSBs. PARP1 may cooperate with the MRN complex to facilitate signaling of DSBs. CtIP (or Ctp1) is another protein that has recently been linked with MRN (Figure 2Figure 2C). It is a mammalian tumor suppressor whose presence in the nucleus is limited to the S and G2 phases of the cell cycle. A recent study indicates that CtIP can form a complex with MRN, directly interacting with Nbs1 in a cell-cycle-dependent manner. The formation of this complex, which also includes BRCA1, requires cyclin-dependent kinase activity. Recent findings indicate that this Brca1–MRN–CtIP complex is important for facilitating DSB resection, which generates the 3′ overhanging single-stranded DNA that is needed both for HR-mediated DSB repair and for the maintenance of checkpoint signaling.What else is left to be examined? Much has been discovered concerning the highly pleiotrophic functions of the MRN complex and new findings are continuously adding complexity. The detailed mechanistic understanding of how MRN really works in vivo remains elusive, however. The real challenge for the future is the integration of all the recent discoveries into mechanism. Therefore much remains to be done and no doubt there are several surprising discoveries still to be made.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases., Author Summary Human cells activate the DNA damage response (DDR) to repair DNA damage and to prevent cells with DNA damage from proliferating. Alterations to the DDR are strongly implicated in the development of cancer. Using the budding yeast model system, we have studied how the regulation of the key DDR component Rad9 is integrated into cell cycle control. The cyclin-dependent kinase Cdc28 that regulates the yeast cell cycle also extensively phosphorylates Rad9 during cell cycle progression. We show here that Cdc28 controls Rad9 function in the activation of the important downstream DNA damage effector kinase Chk1. Two sites of phosphorylation in the N-terminus of Rad9 are crucial for the physical interaction between Rad9 and Chk1 regulated by Cdc28. We propose a novel model for Chk1 activation whereby a subset of Rad9 and Chk1 interacts constitutively in the absence of DNA damage. The Rad9/Chk1 complex is recruited to sites of DNA damage where activation of Chk1 involves additional DDR–specific protein kinases. Human cells contain multiple Rad9-like proteins that are also known to be cell cycle phosphorylated in the absence of exogenous DNA damage, suggesting that our observations may have important implications for DDR regulation in human cells.