Rad53 Activation Research Articles

CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling.

Mechanisms of DNA damage and repair signaling are not completely understood that hinder the efficiency of cancer therapy. Urokinase-type plasminogen activator receptor (PLAUR) is highly expressed in most solid cancers and serves as a marker of poor prognosis. We show that PLAUR actively promotes DNA repair in cancer cells. On the contrary, downregulation of PLAUR expression results in delayed DNA repair. We found PLAUR to be essential for activation of Checkpoint kinase 1 (CHK1); maintenance of cell cycle arrest after DNA damage in a TP53-dependent manner; expression, nuclear import and recruitment to DNA-damage foci of RAD51 recombinase, the principal protein involved in the homologous recombination repair pathway. Underlying mechanism implies auto-/paracrine signaling of PLAUR/TLR4 receptor complex leading to activation of CHK1 and DNA repair. The signaling is induced by a danger molecule released by DNA-damaged cells and mediates, at least partially, activation of DNA-damage response. This study describes a new mechanism of DNA repair activation initiated by auto-/paracrine signaling of membrane receptors PLAUR/TLR4. It adds to the understanding of role of PLAUR in cancer and provides a rationale for therapeutic targeting of PLAUR/TLR4 interaction in TP53-positive cancers.

Correction: Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

[This corrects the article DOI: 10.1371/journal.pgen.1006226.].

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Roleof the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5–SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5–SFR1 and RAD51. We found that SWI5–SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5–SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51–SWI5–SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51.

RAD51 is the central protein in homologous recombination (HR) DNA repair and represents a therapeutic target in oncology. Herein we report a novel class of RAD51 inhibitors that were identified by high throughput screening. In contrast to many previously reported RAD51 inhibitors, our lead compound 1 is capable of blocking RAD51-mediated D-loop formation (IC50 21.3 ± 7.8 μM) at concentrations that do not influence RAD51 binding to ssDNA. In human cells, 1 inhibits HR (IC50 13.1 ± 1.6 μM) without blocking RAD51's ability to assemble into subnuclear foci at sites of DNA damage. We determined that the active constituent of 1 is actually an oxidized derivative (termed RI(dl)-1 or 8) of the original screening compound. Our SAR campaign also yielded RI(dl)-2 (hereafter termed 9h), which effectively blocks RAD51's D-loop activity in biochemical systems (IC50 11.1 ± 1.3 μM) and inhibits HR activity in human cells (IC50 3.0 ± 1.8 μM).

Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication.

In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high-resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD51 paralogue was discovered in Caenorhabditis elegans, and its in vitro characterization has demonstrated a new function for the worm RAD51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts.

Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity.

The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.

Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase.

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.

Abstract 3478: A multi-orthogonal strategy for targeting EGFRvIII expressing glioblastomas

Abstract Epidermal Growth Factor Receptor variant III (EGFRvIII) is a recurrent oncogenic EGFR isoform found in 25-64% of glioblastomas, the most common form of primary brain cancer. While EGFRvIII expressing glioblastomas are initially sensitive to EGFR inhibition, emergence of resistance clones is universal. We previously demonstrated that EGFRvIII expressing glioblastoma can be ablated through inhibition of compensatory mechanisms that allow cancer cells to adapt to EGFRvIII induced oncogenic stress. Here we propose that this strategy can be combined with direct inhibition of EGFR signaling. A synthetic lethal RNAi screen revealed synthetic lethality between Polo-Like Kinase 1 (PLK1) inhibition and EGFRvIII expression in glioblastoma cells. PLK1 mediated glioblastoma adaptation to excessive DNA damage associated with EGFRvIII expression through activation of Rad51, a protein essential for homologous recombination (HR). PLK1 inhibition (by BI2536) enhanced the cytotoxic effects of the DNA damaging agent, temozolomide (TMZ), a DNA alkylating agent and the standard-of-care chemotherapy for glioblastoma. This TMZ-sensitizing effect is 3-5 fold more pronounced in a formed Ink4a/Arf(-/-) EGFRvIII GEMM (genetically engineerd murine model)-derived glioblastoma model relative to an Ink4a/Arf(-/-) PDGF-β GEMM-derived glioblastoma model and correlated to the endogenous level of DNA damage accumulation in these models. Importantly, the tumoricidal and TMZ-sensitizing effects of BI2536 were uniformly observed across Ink4a/Arf(-/-) EGFRvIII glioblastoma clones that acquired independent resistance mechanisms to EGFR inhibitors, Gefitinib and Erlotinib, suggesting these resistant clones retain oncogenic stress that required PLK1 compensation. Although BI2536 significantly augmented the anti-neoplastic effect of EGFR inhibitors in the Ink4a/Arf(-/-) EGFRvIII model, complete ablation was not achieved until TMZ was added. Such synergistic effect was not observed in the Ink4a/Arf(-/-) PDGF-β model. Our study revealed a novel synthetic lethal interaction between PLK1 inhibition and EGFRvIII expression. Our results suggest that optimal therapeutic effect against EGFRvIII expressing glioblastomas requires “multi-orthogonal” integration of oncogenic inhibition, oncogenic stress, and conventional chemotherapy, tailored to the molecular physiology associated with the target cancer genome. Citation Format: Ying Shen, Jie Li, Frank B Furnari, Arshad Desai, Hong-Zhuan Chen, Clark Chen. A multi-orthogonal strategy for targeting EGFRvIII expressing glioblastomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3478. doi:10.1158/1538-7445.AM2015-3478

Abstract A33: γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma

Abstract Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC. Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay. Results: γH2AX expression was significantly higher (p<0.0001) in smokers when compared to non-smokers. Overall survival of patients with high γH2AX expression in their primary tumor was associated with significantly decreased median survival (11 months) when compared to low γH2AX expression (36 months, p=0.0007). HPDE6 cells exposed to CS or NNK, showed more than 80% survival. The surviving cells had altered morphology, acquired mesenchymal characteristics and displayed increased tumorigenicity suggesting that these cells acquire resistance to apoptosis after CS or NNK exposure. In addition, exposure of cells to CS or NNK resulted in generation of γH2AX and induction of phospho-CHK2 expression. RAD51 and phospho-CHK2 co-localize with γH2AX after DNA damage with CS or NNK exposure. Inhibition of CHK2 reduced expression of γH2AX. The product of the tumor suppressor gene BRCA2 co-localized with γH2AX indicating that γH2AX is necessary for the recruitment of DNA repair factors to the site of DNA damage. BRCA2 mutant cell line CAPAN1 exhibited a substantially reduced ability to form co-localization with RAD51 and γH2AX, consistent with a role of γH2AX in DNA damage repair. Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC. Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A33.

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.

Dampening Checkpoint Signaling via Coordinated Action of DNA Repair Scaffolds and the Pph3 Phosphatase

During DNA replication, replication forks are prone to stall and collapse. To prevent genomic instability, DNA damage checkpoint (DDC) kinases are activated and coordinate various cellular responses. In S. cerevisiae, the key DDC kinase Rad53 plays crucial roles in the regulation of transcription, dNTP levels, cell cycle progression and origin firing. Importantly, hyperactivation of Rad53 is deleterious and results in sensitivity to replication stress, indicating the importance for regulating Rad53 activity. Recently, we uncovered a phosphatase-independent mechanism for down-regulating Rad53 signaling named DAMP (Dampens Adaptor Mediated-Phosphosignaling). DAMP relies on the Slx4 scaffold protein competing with the checkpoint adaptor Rad9 at sites of lesions to counteract Rad53 activation. Here we show that DAMP functions in parallel with canonical phosphatase mechanisms for Rad53 down-regulation. We show that slx4Δ cells phenocopy cells lacking the main Rad53 phosphatase, Pph3. Both cells show selective sensitivity to methyl methanesulfonate (MMS), accumulate chromosomal defects and hyperactivate Rad53. Both Slx4 and Pph3 seem to converge to the regulation of the Mus81 nuclease, which is necessary for downstream repair. Interestingly, deletion of both SLX4 and PPH3 leads to a synergistic increase in MMS sensitivity and Rad53 activation, suggesting that efficient down-regulation of DDC signaling requires the coordination of DAMP and phosphatases. We propose that these mechanisms operate in distinct spatio-temporal modes, with Slx4 dampening Rad53 activation at lesions, and Pph3 functioning on free, active pools of Rad53 to turn off the checkpoint response.

Role of Yeast Rad51 Interface Residue F187A and DNA Binding Loop 1 Residue F290 in Coordinating DNA Binding, ATPase, and DNA Strand Exchange Activities

The recombinase Rad51 promotes homologous recombination in eukaryotes. Recombination is initiated when Rad51 forms a presynaptic filament on ssDNA. Filament formation activates the enzymatic activities of Rad51 including ssDNA-dependent ATP hydrolysis, homology search and DNA strand exchange. Proper filament assembly is crucial for double-strand break repair and the avoidance of cancer. Previous studies showed that the yeast Rad51 H352 residue, located at the protomer-protomer interface near the ATPase site, is important for allosteric communication. The H352Y variant exhibits increased affinity for ssDNA but is unable to undergo multiple turnovers of ATP hydrolysis. The crystal structure of H352Y shows that F187, another conserved residue at the interface, may also be a significant player. In this study, we show that the F187A variant can hydrolyze ATP but has a decreased affinity for DNA and thus a deficiency in strand exchange activity is seen. Additionally, we report a novel X-ray crystal structure of yeast Rad51 resolving the DNA binding loop 1. This structure reveals a conserved aromatic residue, F290 that contacts the residues of the ATPase site. Like wild-type Rad51, the F290A variant possess ATP-dependent binding to both dsDNA and ssDNA. Unlike wild-type, however, F290A is not active for ATPase and DNA strand exchange activities. Data suggest that the deficient strand exchange activities for both variants is due to the disruption of the signaling pathway between the binding sites for ATP and DNA. Funded by NIH.

Phosphorylation of Sae2 Mediates Forkhead-associated (FHA) Domain-specific Interaction and Regulates Its DNA Repair Function

Saccharomyces cerevisiae Sae2 and its ortholog CtIP in higher eukaryotes have a conserved role in the initial processing of DNA lesions and influencing their subsequent repair pathways. Sae2 is phosphorylated by the ATR/ATM family kinases Mec1 and Tel1 in response to DNA damage. Among the Mec1/Tel1 consensus phosphorylation sites of Sae2, we found that mutations of Thr-90 and Thr-279 of Sae2 into alanine caused a persistent Rad53 activation in response to a transient DNA damage, similar to the loss of Sae2. To gain insight into the function of this phosphorylation of Sae2, we performed a quantitative proteomics analysis to identify its associated proteins. We found that phosphorylation of Thr-90 of Sae2 mediates its interaction with Rad53, Dun1, Xrs2, Dma1, and Dma2, whereas Rad53 and Dun1 additionally interact with phosphorylated Thr-279 of Sae2. Mutations of the ligand-binding residues of Forkhead-associated (FHA) domains of Rad53, Dun1, Xrs2, Dma1, and Dma2 abolished their interactions with Sae2, revealing the involvement of FHA-specific interactions. Mutations of Thr-90 and Thr-279 of Sae2 caused a synergistic defect when combined with sgs1Δ and exo1Δ and elevated gross chromosomal rearrangements. Likewise, mutations of RAD53 and DUN1 caused a synthetic growth defect with sgs1Δ and elevated gross chromosomal rearrangements. These findings suggest that threonine-specific phosphorylation of Sae2 by Mec1 and Tel1 contributes to DNA repair and genome maintenance via its interactions with Rad53 and Dun1.

The In Vitro Characterization of the Role of Rad57 in Rad51 Catalyzed Homologous Recombination Repair

The homologous recombination of DNA is necessary for the accurate repair of double-strand DNA breaks. The activity of this repair pathway must remain at an appropriate level; repair must effectively recover stalled or broken replication forks, without leading to genomic instability and cancer. This balance requires precise control over these reactions. A number of proteins involved in DNA repair mechanisms have been identified; some have been studied extensively, and their functions and biochemical roles characterized. Other proteins have yet to be studied, and their role in recombination remains unclear. The Rad57 protein has been identified as a RA51 paralog and is thought to play a role in regulating Rad51 activities in homologous recombination. However, to date few studies have focused on specifically analyzing the biochemical functions of Rad57. To better understand the functions of Rad57 in homologous recombination, we have cloned, expressed, purified Rad57 and characterized its biochemical activities surrounding homologous recombination. The characterized activities of Rad57 include its DNA binding properties and its effect on Rad51 catalyzed actions.

Interplay between histone H3 lysine 56 deacetylation and chromatin modifiers in response to DNA damage.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage.

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase.

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

P14 - Glutathione is essential to preserve nuclear function and cell survival under oxidative stress

Organisms growing in aerobic environments must cope with Reactive Oxygen Species (ROS). Although ROS damage all the cellular macromolecules, they play a central role in a range of biological processes requiring a tight control of redox homeostasis. It is achieved by antioxidant systems involving a large collection of enzymes that scavenge or degrade the ROS produced endogenously during cell growth. In addition to this enzymatic protection against ROS, cells also contain small antioxidant molecules, such as glutathione (GSH). With an intracellular concentration between 1 and 10mM, GSH is the most abundant non-protein thiol in the cell and is considered as the major redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing different amounts of GSH.We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear DNA and nuclear functions are protected against oxidative injury, as exemplified by low mutation frequency, moderate histone carbonylation, activation of the checkpoint kinase Rad53 and of the H2O2 transcriptional response.We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Abstract SY33-01: Mechanism of tumor suppression by the Fanconi anemia proteins

Abstract DNA Interstrand crosslink (ICL) repair takes place at sites where the two strands of the DNA have become covalently linked by the byproducts of lipid peroxidation, endogenous aldehydes, and yet unknown metabolites. The repair of these lethal lesions takes place during DNA replication and requires a dual excision of the crosslinked bases and repair of the resulting double-strand breaks. This feat is accomplished in a multi-step process mediated by the Fanconi anemia (FA) pathway and factors that promote Homologous Recombination (HR), including BRCA1 and BRCA2 (reviewed in (1)). FA patients lack components of this pathway and suffer from bone marrow failure and infertility presumably due to the inability to maintain hematopoietic and germline stem cells. FA is also associated with a very high incidence of cancer, most likely due to the mutagenic nature of incompletely repaired ICLs. On the other hand, the induction of ICL is a major method of cancer treatment and often leads to excellent outcomes (e.g. cisplatin treatment of testicular cancer). Major effort has been directed into the identification of all of the components of the ICL repair pathways and the elucidation of the mechanism of such repair in the hopes of understanding how it suppresses tumorigenesis. Fanconi anemia registry (IFAR) housed at the Rockefeller University has been collecting patient samples and information since 1982. Using candidate gene sequencing of patient samples from the IFAR, we first found SLX4 as a new FA gene. SLX4 is a scaffold protein that interacts with three nucleases: XPF, MUS81, and SLX1 (2-4). We showed that this “Swiss army knife of repair” is mutated in patients with typical features of Fanconi anemia (5). Using SLX4 null patient cell lines, we explored the nuclease requirements in different DNA repair pathways during DNA replication and showed that the SLX4-bound XPF nuclease is essential for the ICL repair (6). This has been recently validated by work that identified XPF as a gene mutated in FA (7). In a separate study, we demonstrated that SLX4 is an important regulator of processing of replication intermediates including the Holliday junctions even under conditions of unperturbed DNA replication (8). This activity, although important for normal cellular growth, can be very mutagenic and thus cancer inducing. In our newest work, we have concentrated on a 12-year-old patient who was born with radial dysplasia, absent right thumb and tethered cord; the patient also exhibited enuresis and several absent permanent teeth. Marrow function is normal to date. No mutations in any of the known FA genes were identified. Whole exome sequencing identified an unexpected novel de novo mutation in RAD51. RAD51 is the mammalian homolog of RecA, mediating the homology search during HR. Although RAD51 foci formation is reduced and delayed following treatment with a range of DNA damaging agents, the patient fibroblasts do not exhibit sensitivity to ionizing radiation. In addition, the patient fibroblasts show wild type levels of sister chromatid exchanges following mitomycin C treatment, and are capable of performing HR for repairing dysfunctional GFP gene, as assessed by the DR-GFP assay. Collectively, these results suggest HR repair of double strand breaks is not impaired in the patient cells. Patient fibroblasts are hypersensitive to crosslinking agents and PARP inhibition. Following mitomycin C treatment, the cells exhibit elevated levels of both RPA foci formation and phosphorylation, indicating that RAD51 inhibits resection of nascent DNA strands, thereby stabilizing the ICL for its subsequent repair. Our study of the RAD51 patient mutation identifies the essential function of RAD51 at sites of stalled replication forks that is critical for the accurate repair of ICLs. Our results also suggest that the tumor suppressive role of RAD51-interacting proteins, including BRCA2, RAD51C, and RAD51D (all implicated in breast and ovarian cancer development) may exert their effect through this stabilizing activity of RAD51 at stalled replication forks.