DNA Damage Repair Proteins Research Articles

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.

Recruitment and retention dynamics of RECQL5 at DNA double strand break sites

RECQL5 is one of the five human RecQ helicases, involved in the maintenance of genomic integrity. While much insight has been gained into the function of the Werner (WRN) and Bloom syndrome proteins (BLM), little is known about RECQL5. We have analyzed the recruitment and retention dynamics of RECQL5 at laser-induced DNA double strand breaks (DSBs) relative to other human RecQ helicases. RECQL5-depleted cells accumulate persistent 53BP1 foci followed by γ-irradiation, indicating a potential role of RECQL5 in the processing of DSBs. Real time imaging of live cells using confocal laser microscopy shows that RECQL5 is recruited early to laser-induced DSBs and remains for a shorter duration than BLM and WRN, but persist longer than RECQL4. These studies illustrate the differential involvement of RecQ helicases in the DSB repair process. Mapping of domains within RECQL5 that are necessary for recruitment to DSBs revealed that both the helicase and KIX domains are required for DNA damage recognition and stable association of RECQL5 to the DSB sites. Previous studies have shown that MRE11 is essential for the recruitment of RECQL5 to the DSB sites. Here we show that the recruitment of RECQL5 does not depend on the exonuclease activity of MRE11 or on active transcription by RNA polymerase II, one of the prominent interacting partners of RECQL5. Also, the recruitment of RECQL5 to laser-induced damage sites is independent of the presence of other DNA damage signaling and repair proteins BLM, WRN and ATM.

Abstract LB-409: Network analysis of RNA-seq data comparing triple negative breast cancer to normal breast tissues

Abstract Introduction: Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to other breast cancers. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. Using next-generation RNA-seq, we embarked on a study to compare the transcriptomes of TNBC and normal breast to comprehensively identify dysregulated networks, pathways, transcription factors, and high connectivity drug targets that can be exploited for therapeutic discovery. Methods: cDNA libraries from 10 normal breast tissues from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center and 10 TNBC tumors were sequenced on an Applied Biosystems (ABI) SOLiD3 sequencer. Mapping of reads to the human genome (hg18) was performed using ABI BioScope 1.2; differential gene expression was analyzed using Partek Genomics Suite; and network, pathway, and transcription factor analysis was performed using Ingenuity Systems iReport and IPA 9.0. Results: Canonical pathway analysis revealed highly elevated and enriched expression in BRCA/DNA repair pathways, PI3K/AKT, and integrin signaling. Particularly, targeting BRCA/DNA repair pathways with PARP inhibitors has demonstrated some clinical success. Our pathway analysis reveals additional targets: ATM, CHK1/2, & PLK1 that are highly overexpressed and are integral to the BRCA pathway. To determine key regulators of our dataset, we employed IPA's transcription factor (TF) analysis that utilizes a literature knowledge base to identify TFs that explain a set of differential expression data, and classifies them as either activated or inhibited. We observed that TP53, RB1, & CDKN2A were significantly inhibited. This is congruent with current literature showing that TP53 & RB1 as the most predominantly mutated genes in TNBC. In terms of activation, TBX2 and c-MYC were significantly activated in our dataset, suggesting a highly pro-tumorigenic transcriptional programme. In addition, RNA-seq detects pre-miRNAs. When integrated into the analysis, the top ranked network reveals a large set of dysregulated miRNAs secondary to inhibited TP53. When looking at druggable kinases, we performed a connectivity analysis to determine major hub proteins. Our top three kinases were SRC, IKBKE, and AKT1. Of note AKT inhibitors have recently entered early phase clinical trials in breast cancer. Conclusions: Network analysis reveals a concerted programme of proteins and transcription factors that drive a highly pro-tumorigenic phenotype revolving around nonfunctional TP53, and upregulated PI3K/AKT & DNA damage repair pathways. The analysis reveals additional DNA damage repair proteins in addition to PARP and highly connected kinases as potential drug targets for TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-409. doi:1538-7445.AM2012-LB-409

Activation of the DNA Damage Response Pathway and the Role of NBS1 in Response to Hedgehog Signaling Inhibition

Hedgehog (HH) signaling actively regulates key processes such as cell differentiation and cell proliferation during embryonic development. Aberrant HH pathway activation has been identified in several human cancers, however its role in colon carcinoma remains unclear. The GLI proteins downstream of signaling via PTCH and SMO transcriptionally regulate HH target genes. Pharmacologic inhibition of GLI1 and GLI2 by GANT61, or by a C‐terminus deleted GLI3 repressor, GLI3R, induced DNA damage (marked by increased γH2AX, p‐ATM and p‐Chk2) and extensive cell death in human colon carcinoma cells. Exposure to GANT61 for 24 hr followed by washout allowed DNA repair and reversal of cytotoxicity. NBS1 is a key regulator in intra‐S‐phase checkpoint activation, DNA damage signaling, and DNA repair. Following GLI1/GLI2 inhibition, NBS1 nuclear foci colocalized in single cells with MDC1 but not with γH2AX foci, both engaged in recruitment of DNA damage signaling and repair proteins to DNA break sites. During DNA damage, NBS1 demonstrated limited binding to chromatin, while γH2AX and MDC1 binding was induced and sustained. During DNA repair NBS1 and MDC1 were highly chromatin‐bound while γH2AX was released from chromatin. These findings provide new insight into the critical role of GLI1/GLI2 in driving cellular survival in colon cancer and mechanisms that follow dysregulation of GLI1/GLI2 function that determine cell death.

Enhanced cytotoxicity of PARP inhibition in mantle cell lymphoma harbouring mutations in both ATM and p53

Poly-ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia-telangiectasia mutated (ATM)-deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM-deficient MCL cells, olaparib induced DNA-PK-dependent phosphorylation and stabilization of p53 as well as expression of p53-responsive cell cycle checkpoint regulators, and inhibition of DNA-PK reduced the toxicity of olaparib in ATM-deficient MCL cells. Thus, both DNA-PK and p53 regulate the response of ATM-deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53-deficient malignancies.

Bortezomib-induced “BRCAness” sensitizes multiple myeloma cells to PARP inhibitors

AbstractChromosomal instability is a defining feature of clonal myeloma plasma cells that results in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis, the ubiquitin-proteasome system is also involved in the regulation of DNA damage-repair proteins. In the present study, we show that proteasome inhibition induces a “BRCAness” state in myeloma cells (MM), with depletion of their nuclear pool of ubiquitin and abrogation of H2AX polyubiquitylation, an essential step for the recruitment of BRCA1 and RAD51 to the sites of DNA double-stranded breaks (DSBs) and the initiation of homologous recombination (HR)–mediated DNA repair. Inhibition of poly-ADP-ribose-polymerase 1 and 2 (PARP1/2) with ABT-888 induced transient DNA DSBs that were rapidly resolved and thus had no effect on viability of the MM cells. In contrast, cotreatment of MM cell lines and primary CD138+ cells with bortezomib and ABT-888 resulted in the sustained accumulation of unrepaired DNA DSBs with persistence of unubiquitylated γH2AX foci, lack of recruitment of BRCA1 and RAD51, and ensuing MM-cell death. The heightened cytotoxicity of ABT-888 in combination with bortezomib compared with either drug alone was also confirmed in MM xenografts in SCID mice. Our studies indicate that bortezomib impairs HR in MM and results in a contextual synthetic lethality when combined with PARP inhibitors.

Bortezomib Impairs Myeloma Cells (MM) Homologous Recombination Through Inhibition of the E2-Ubiquitin-Conjugating Enzyme UBC13

Abstract 1834 Background:Chromosomal instability is a defining feature of clonal MM plasma cells resulting in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis the ubiquitin-proteasome system is also involved in the regulation of DNA damage repair proteins. We have recently reported that proteasome inhibition induces a BRCAness state in MM cells (MM) resulting in a contextual synthetic lethality when combined with PARP inhibitors. We now report on the mechanisms by which bortezomib impairs homologous recombination (HR) mediated repair of DNA double stranded breaks (DNA-DSBs). Methods and results:Using the DR-GFP/SceI reporter assay in which cells are transfected with a plasmid tandemly expressing mutated GFP genes (5’-Sce/GFP – truncated IGFP-3’) and followed by infection with an adenovirus (AdNGUS24i) expressing the yeast Sce endonuclease, we have confirmed that treatment with bortezomib does severely impair HR in MM cells. At the transcriptional level, proteasome inhibition transiently (6–12 hours post treatment) reduced the mRNA expression of several HR and DNA damage repair genes (BRCA1, BRCA2, RAD51 and FANCD2) followed by recovery to baseline levels by 24 hours. This transient downregulation of these DNA damage repair genes was also confirmed in a promoter luciferase reporter screen. At the post-trancriptional level, examining the dynamics and kinetics of DNA-DSBs repair induced by radiation therapy or PARP inhibition, we have demonstrated that bortezomib does not alter the initial phase of DNA damage sensing (MRN complex recruitment, ATM and H2AX phosphorylation and MDC1 activation) but does impair the Lys63-poly-ubiquitylation of histones γH2AX and H2A. This histone ubiquitylation is a required modification for the second wave of DNA repair proteins recruitment and retention of BRCA1 and RAD51 at the sites of DNA-DSBs. Furthermore, we have shown that bortezomib treatment depleted the nuclear pools of ubiquitin and abrogated the poly-ubiquitylation (lack of co-localization poly-Ub FK2 foci with γH2AX), but not the phosphorylation and foci formation of histone H2AX. Therefore, bortezomib appears to impair histones (H2AX and H2A) poly-ubiquitylation, a process that is dependent on the activation and recruitment of the E3 ubiquitin-protein ligases RNF8 and RNF-168 as well as the ubiquitin-E2 conjugating enzyme UBC13 to the sites of DNA-DSBs. Western blot analysis of the nuclear and cytoplasmic fractions of cellular extracts, revealed that bortezomib dramatically reduced the nuclear accumulation of UBC13 in response to DNA-DSBs as well as the formation of UBC13-Ubiquitin thiolester bonds formation and hence impaired the transfer of ubiquitin to nucleosomal histones. Conclusion:Our studies demonstrate that proteasome inhibition with bortezomib severely impairs homology-mediated repair of DNA breaks in MM cells at the post-transcriptional level by altering the nuclear accumulation and function of the E2-ubiquitin conjugating enzyme UBC13. These results explain the observed clinical synergy between bortezomib and several DNA damaging agents and support further clinical investigation of the combination of these class of drugs in MM patients. Disclosures:Neri:Celgene: Honoraria, Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.

728 BRCA1 induces cardiac protection through a P53-dependent pathway

Recent data implicate the DNA-damage repair protein, BRCA1, as an essential regulator of cardiac structure and function. We hypothesized that BRCA1 regulates p53-mediated apoptotic cell death in cardiomyocytes and through this mechanism attenuates cardiac failure.

DNA-damage response and repair activities at uncapped telomeres depend on RNF8

Loss of telomere protection causes natural chromosome ends to become recognized by DNA-damage response and repair proteins. These events result in ligation of chromosome ends with dysfunctional telomeres, thereby causing chromosomal aberrations on cell division. The control of these potentially dangerous events at deprotected chromosome ends with their unique telomeric chromatin configuration is poorly understood. In particular, it is unknown to what extent bulky modification of telomeric chromatin is involved. Here we show that uncapped telomeres accumulate ubiquitylated histone H2A in a manner dependent on the E3 ligase RNF8. The ability of RNF8 to ubiquitylate telomeric chromatin is associated with its capacity to facilitate accumulation of both 53BP1 and phospho-ATM at uncapped telomeres and to promote non-homologous end-joining of deprotected chromosome ends. In line with the detrimental effect of RNF8 on uncapped telomeres, depletion of RNF8, as well as of the E3 ligase RNF168, reduces telomere-induced genome instability. This indicates that, besides suppressing tumorigenesis by mediating repair of DNA double-strand breaks, RNF8 and RNF168 might enhance cancer development by aggravating telomere-induced genome instability.

Valproic acid synergistically enhances the cytotoxicity of gossypol in DU145 prostate cancer cells: An iTRTAQ-based quantitative proteomic analysis

Gossypol (GOS), a BH3 mimetic, has been investigated as a sensitizing co-therapy to radiation and chemotherapy in treatment of metastatic prostate cancer. In this study, we found that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), counteracted the suppressive effect of GOS on histone H3 acetylation and enhanced the cytotoxicity of GOS to DU145 prostate cancer cells. Significant synergistic effects were observed in combined GOS and VPA treatment, culminating in more DNA damage and cell death. The iTRAQ-based quantitative proteomic analysis revealed differential proteomic profiles in cells treated with VPA, GOS or their combination. In GOS-treated cells, oxidative phosphorylation-related proteins were depressed and endoplasmic reticulum stress markers were upregulated. In the presence of VPA, the GOS-induced mitochondrial stress was further enhanced since glycolysis- and hypoxia-associated proteins were upregulated, suggesting a disruption of energy metabolism in these cells. Furthermore, the DNA damage repair ability of cells co-treated with GOS and VPA was also decreased, as evidenced by the downregulation of DNA damage repair proteins and the enhancement of DNA fragmentation and cell death. These findings suggest that GOS in combination with an HDACI has the potential to increase its clinical efficacy in the treatment of prostate cancer.

Abstract 2476: Acetylated Survivin functions in DNA damage repair

Abstract Survivin is an oncogenic protein that participates in cell division and inhibits apoptosis in cancer cells. These functions are dependent on its interaction with partner proteins within distinct subcellular compartments. Survivin is highly expressed in malignant tumors yet minimally expressed in normal tissues, making it an attractive target for molecular therapy. Inhibition of Survivin function enhances tumor cell sensitivity to radiation, the mechanism of which has been reported to result from a disruption of DNA-double-strand break repair processes. Recently, we demonstrated that Survivin is post-translationally modified by acetylation at numerous lysine residues, one of which is responsible for its subcellular transport. Acetylation at Lys-129 facilitates Survivin homodimerization which prevents its binding to the CRM1 nuclear export protein, inhibiting its translocation from the nucleus to the cytoplasm. The object of this study was to determine the potential relationship between Survivin acetylation and its function in nuclear repair processes. To elucidate the association between Survivin acetylation, nuclear localization and DNA double-strand break repair we subjected HeLa cells to 4Gy γXRT and examined Survivin acetylation at serial time points (20, 40, 60 min) after irradiation. An immediate deacetylation of Survivin was observed at 20 minutes, followed by an increase in both global acetylation and acetylation at Lys-129 at 40 minutes. The increase in Lys-129 acetylation correlated with its nuclear accumulation, as demonstrated by indirect immunofluorescence staining and immunoblotting after subcellular fractionation. Acetylated Survivin was found in endogenous immunoprecipitants with the DNA damage repair protein, Ku70, following γ-irradiation. Indirect immunoflourescence revealed co-localization of Ku70 and acetylated Survivin within the nucleus at similar time points after irradiation suggesting that Survivin acetylation may be required for Ku70 complex formation within the nucleus. These results implicate acetylated Survivin as a regulator of DNA damage repair through the Ku70 DNA repair pathway and further our understanding of Survivin function within the nuclear compartment of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2011-2476

Abstract 976: Hypoxic microenvironment promotes breast cancer stem cells through regulation of Polycomb protein expression

Abstract The microenvironment of solid tumors contains regions of poor oxygenation as a result of hypoxia, which is associated with high grade breast cancer and poor prognosis. However, critical regulatory mechanism linking tumor microenvironment to cancer progression remains unclear. Cancer stem cells (tumor initiating cells) are suggested to be responsible for initiation and aggressive progression of cancer. It is recently proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genomic/genetic mutations and drives cancer progression. Additionally, high expression of a Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to aggressive progression of breast cancer. Here, we discovered that hypoxic microenvironment upregulates HIF1α-mediated transcriptional activation of EZH2 in CD44+CD24−/low breast tumor initiating cells (BTICs) isolated from several human primary tumor samples. Enhanced EZH2 expression was required for the hypoxia-promoted BTICs, where hypoxia significantly increased BTIC abundance, tumor sphere formation and tumor growth in mammary fat pads implanted with less than 500 primary BTICs. Using cDNA expression array and genome wide SNP array analysis, we further identified a novel mechanism in which enhanced expression of EZH2 by hypoxia downregulates DNA damage repair proteins through H3K27 methylation and thereby allows the acquisition of recurrent genomic abnormalities such as RAF1 gene amplification in BTICs. Using proteomic analysis, we demonstrated that RAF1 amplification highly activates its downstream ERK-β-catenin signaling, which leads to enhanced survival and proliferation of the BTICs. Furthermore, using immunohistochemistry analysis in a cohort of breast tumor samples, we found that EZH2 expression is positively correlated with RAF1, p-ERK and β-catenin expression in the high grade, poorly-differentiated tumors but not in the low grade, well-differentiated tumors, suggesting EZH2-amplified RAF1-ERK-β-catenin signaling may contribute significantly to promote BTICs and aggravate breast cancer malignancy. Our data also reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, can significantly suppress EZH2-promoted BTICs. To the best of our knowledge, this is the first study that provides evidences linking a specific genomic aberration mediated by EZH2-impaired DNA damage response to expansion of BTICs. Together, this study elucidates a previously unidentified therapeutic effect of RAF1-ERK signaling inhibitors to prevent breast cancer progression by eliminating BTICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 976. doi:10.1158/1538-7445.AM2011-976

DNA Repair Proteins Are Directly Involved in Regulation of Gene Expression during Plant Immune Response

Systemic acquired resistance (SAR), an inducible plant-defense response to local infection, requires the signaling molecule salicylic acid (SA) and the transcriptional coactivator NPR1, with concerted activation of pathogenesis-related (PR) genes. Arabidopsis sni1 is an npr1 suppressor and derepression of defense genes in sni1 causes reduced growth and fertility and increased homologous recombination. Characterizing suppressors of sni1, we identify the DNA damage repair proteins SSN2 and RAD51D as genetic and physical interactors with SNI1. During plant defense, SSN2 and possibly RAD51D replace the transcription repressor SNI1 at pathogenesis-related gene promoters. In the presence of SNI1, NPR1 is also required for SSN2 binding. Thus, coordinated action of SNI1, SSN2-RAD51D, and NPR1 ensures the tight control of plant immune gene expression. Given that the SSN2-RAD51D complex is conserved in eukaryotes, their dual function in homologous recombination and transcription regulation of plant-defense genes suggests a general link between these two stress responses.

EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-β-Catenin Signaling

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-β-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.

UHRF1 confers radioresistance to human breast cancer cells

Purpose: To investigate the effect of ubiquitin-like with plant homeodomain (PHD) and ring finger domains 1 (UHRF1) overexpression on radiosensitivity to X-rays in human breast cancer MDA-MB-231 cells.Materials and methods: Cell survival was determined by colony formation assay; cell cycle distribution was measured by flow cytometry; apoptosis was evaluated by DNA fragmentation assay and Annexin V apoptosis detection kit; protein expression was analysed by Western blot assay; chromosome aberrations (centric rings and dicentrics) were assayed by conventional chromosome analysis.Results: A significant decrease of radiosensitivity to X-rays was observed in MDA-MB-231 cells transfected with a full-length of human UHRF1 cDNA (MDA-MB-231/UHRF1) compared to the control cells (MDA-MB-231/parental and MDA-MB-231/pcDNA3 [mammalian expression vector]), and the similar results were observed in MDA-MB-468 cells. In contrast, a decreased expression of UHRF1 by a specific UHRF1-small interfering RNA (siRNA) significantly enhanced cell radiosensitivity. The UHRF1-mediated radioresistance was correlated with a G2Ra/M arrest, a decreased induction of apoptosis, a down-regulation of the pro-apoptotic protein anti-B cell lymphoma/leukemia 2 (bcl-2) associated X protein (Bax) and a up-regulation of the DNA damage repair proteins Lupus Ku autoantigen protein p70 (Ku-70) and Lupus Ku autoantigen protein p80 (Ku-80). Furthermore, chromosomal aberrations (centric rings and dicentrics) by X-rays were less in MDA-MB-231/UHRF1 than in MDA-MB-231/parental and MDA-MB-231/pcDNA3 control cells.Conclusions: These results suggested that UHRF1 may be a new target in the radiotherapy of breast cancer via affecting apoptosis and DNA damage repair.

Selection and the Cell Cycle: Positive Darwinian Selection in a Well-Known DNA Damage Response Pathway

Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.

MOF and H4 K16 Acetylation Play Important Roles in DNA Damage Repair by Modulating Recruitment of DNA Damage Repair Protein Mdc1

MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G(2)/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process.

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27(kip1) gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27(kip1) transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27(kip1) transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70(-/-) ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 μM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.

New regulators of the BRCA1 response to genotoxic stress

The breast and ovarian predisposition protein BRCA1 is a required component of the mammalian response to double-stranded DNA damage. Its conserved BRCT domains are required for BRCA1 accumulation to sites of repair, while the conserved N-terminal RING domain is able to catalyse the conjugation of ubiquitin and act as an E3 ubiquitin ligase. Disruption of either of these domains by missense mutation is associated with disease development. The SUMO conjugation pathway has been implicated in DNA damage response in model organisms, and in Caenorhabditis elegans the Brac1 binding partner Bard1 associates with the SUMO E2 conjugating enzyme Ubc9. In mammalian cells, BRCA1 has been found to be associated with free SUMO-1 resulting in altered transcription. We undertook to examine the potential influence of the SUMO pathway on BRCA1 response to genotoxic stress. Using a range of biochemical and cell-biology techniques, we have shown that BRCA1 is modified by SUMO in response to genotoxic stress, and co-localises at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO conjugating enzyme Ubc9. PIAS SUMO E3 ligases co-localise with and modulate SUMO modification of BRCA1, and are required for BRCA1 ubiquitin ligase activity in cells. In vitro SUMO modification of the BRCA1:BARD1 heterodimer greatly increases its ligase activity, identifying it as a SUMO regulated ubiquitin ligase. Further, PIAS SUMO ligases are required for complete accumulation of double-strand DNA damage repair proteins subsequent to RNF8 accrual, and for proficient double-strand break repair. Because the two features of BRCA1 activity regulated by the SUMO pathway, ubiquitin ligase activity and accumulation at sites of DNA damage, are also inhibited by some BRCA1 mutations that predispose to breast cancer and ovarian cancer, it seems highly likely that the SUMO pathway will be of relevance to cancer predisposition and development.

Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells

Copper oxide nanoparticles (CuO NPs) are increasingly used in various applications. Recent studies suggest that oxidative stress may be the cause of the cytotoxicity of CuO NPs in mammalian cells. However, little is known about the genotoxicity of CuO NPs following exposure to human cells. This study was undertaken to investigate CuO NPs induced genotoxic response through p53 pathway in human pulmonary epithelial cells (A549). In addition, cytotoxicity and oxidative stress markers were also assessed. Results showed that cell viability was reduced by CuO NPs and degree of reduction was dose dependent. CuO NPs were also found to induce oxidative stress in dose-dependent manner indicated by depletion of glutathione and induction of lipid peroxidation, catalase and superoxide dismutase. The expression of Hsp70, the first tier biomarker of cellular damage was induced by CuO NPs. Further, CuO NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and MSH2 expression. These results demonstrate that CuO NPs possess a genotoxic potential in A549 cells which may be mediated through oxidative stress. Our short-term exposure study of high level induction of genotoxic response of CuO NPs will need to be further investigated to determine whether long-term exposure consequences may exist for CuO NPs application.