Facilitates DNA Damage Repair Research Articles

K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair.

Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys63 (K63) linkage bound to DNA through a motif we called the "DNA-interacting patch" (DIP), which is composed of the adjacent residues Thr9, Lys11, and Glu34 Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile44 patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient-derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.

LINP1 facilitates DNA damage repair through non-homologous end joining (NHEJ) pathway and subsequently decreases the sensitivity of cervical cancer cells to ionizing radiation

ABSTRACTLncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) is an lncRNA which promotes therapeutic resistance in triple-negative breast cancer (TNBC). However, the expression and function of LINP1 in cervical cancer is not yet well-understood. In this study, we evaluated the expression levels of LINP1 in tumor tissues and cell lines of cervical cancer. We found that LINP1 associates with NHEJ proteins (Ku80 and DNA-PKcs). LINP1 translocates from cytosol to nucleus in response to irradiation. In addition, LINP1 knockdown significantly increases the levels of cleaved caspase3 and PARP, leading to enhanced cell apoptosis after ionizing radiation (IR). LINP1-knockdown cells showed delayed repairs of DNA double-strand breaks (DSBs) after IR. Finally, LINP1 knockdown increases radiosensitivity of Hela S3 cells. These results suggest that LINP1 facilitates DSBs repair through NHEJ pathway and may thus serve as a prognostic marker and a potential target for the therapy of cervical cancer.

The protective role of DOT1L in UV-induced melanomagenesis

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.

OxyS small RNA induces cell cycle arrest to allow DNA damage repair

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS‐induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read‐through transcription into a cryptic prophage encoding kilR. The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS‐mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

The Bone Marrow Environment Promotes Resistance to Mitoxantrone and Etoposide By Distinct Mechanisms in Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children’s Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p<0.0001). Results were similar for mitoxantrone treatment. We also found increased DNA damage signaling (%cPARP-/gH2AX+) after mitoxantrone treatment for AML cells in stromal co-culture (34.5 ±3.0% in cells cultured alone, v. 58.6 ±3.1% in HS5 co-cultures, p<0.0001). However, DNA damage signaling was not significantly increased after etoposide treatment. For 5 samples treated with both chemotherapy agents, we also measured pDNA-PKcs and pATM by FACS. Treatment with mitoxantrone, but not etoposide, induced more pDNA-PKcs (MFI, 25.7 ±4.2 untreated, v. 61.7 ±5.1 mitoxantrone, p<0.001, v. 31.5 ±4.8 etoposide) in AML cells cultured alone. However, stroma did not further increase pDNA-PKcs. These results suggest that the NHEJ pathway is important for the repair of DNA damage caused by mitoxantrone, and that stromal cells increase DNA damage signaling by a mechanism not involving increased pDNA-PKcs.To better understand etoposide resistance, 29 of the 44 primary AML samples were cultured on stroma overnight, and activation of intracellular signaling pathways, including pY-STAT3, pY-STAT5, and pERK1/2, was measured by FACS. Responses were heterogeneous overall, but we found several patterns of stroma-induced signaling. For example, a sample that strongly activated a given pathway when co-cultured with HS5 also responded strongly to HS27A cells, and this was particularly true for pY-STAT3 (R=0.71, p<0.0001) and pY-STAT5 (R=0.76, p<0.0001). We also found a correlation between pY-STAT3 and pY-STAT5, such that samples that showed a strong activation of STAT3 with stromal co-culture had a similar activation of STAT5 (R=0.94, p<0.0001, on HS27A). As for signaling pathways in relationship to apoptosis, higher levels of pERK1/2 were associated with lower levels of apoptosis (R=-0.5182, p<0.01, on HS27A), suggesting that ERK1/2 activation may promote resistance to etoposide.In summary, we found that DNA damage signaling is induced in AML cells by mitoxantrone, and it is augmented in cells co-cultured with stroma, likely contributing to mitoxantrone resistance. This mechanism does not occur with etoposide. Instead, we found that stromal environment-induced ERK1/2 signaling may enhance etoposide resistance in pediatric AML patient samples. Further studies to confirm the role of pERK1/2 in stroma-induced etoposide resistance are underway. DisclosuresNo relevant conflicts of interest to declare.

P53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA.

Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5′-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3′→5′ exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil–DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.

The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.

The autotaxin–LPA2 GPCR axis is modulated by γ-irradiation and facilitates DNA damage repair

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)–LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin−Sca-1+c-Kit+ enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.

MicroRNA-138 Regulates DNA Damage Response in Small Cell Lung Cancer Cells by Directly Targeting H2AX

Lung cancer is the leading cause of cancer death worldwide and small cell lung cancer (SCLC) accounts for a significant proportion of all lung cancer cases. Even so, the underlying mechanism governing SCLC development remains poorly understood and SCLC related cancer death stands high despite decades of intensive investigation. We noted that both miR-138 and H2AX have been implicated in development of various malignancies. Also, there is a recent report showing the role of miR-138 in mediating DNA damage response by targeting H2AX. In light of these data, we sought to characterize the role of miR-138 for SCLC cell growth and cell-cycle progression by regulating H2AX expression. Results showed that miR-138 is significantly down-regulated in SCLC tumor tissues as well as in three SCLC cell lines. After successfully engineering miR-138 overexpression in one of the SCLC cell lines, NCI-H2081, we observed a remarkable reduction of cell growth and a significant inhibition on cell-cycle progression. Moreover, we were able to show that miR-138 potently inhibits H2AX expression, which suggests that H2AX may serve as a downstream executor for miR-138. Consistent with this hypothesis, we found that engineered H2AX knockdown achieves a similar effect as observed for miR-138 overexpression in terms of SCLC growth and cell cycle regulation. We also showed that H2AX overexpression largely abolished miR-138-mediated SCLC cancer cell growth and cell-cycle progression inhibition, which strongly suggests, at least in vitro, that miR-138 potently regulates SCLC development by targeting H2AX. In addition, we found lower miR-138 expression confers SCLC cells with greater DNA damage repair capacity. Finally, we were able to show miR-138 overexpression inhibits DNA damage repair in SCLC cells while miR-138 knockdown further facilitates DNA damage repair in these cells after IR. To date, there has been no study showing the role of miR-138/H2AX machinery in SCLC development. Our results may shed a light to development of new lines of SCLC diagnosis and treatment approaches.

The role of poly(ADP-ribosyl)ation in DNA damage response and cancer chemotherapy.

DNA damage is a deleterious threat, but occurs daily in all types of cells. In response to DNA damage, poly(ADP-ribosyl)ation, a unique posttranslational modification, is immediately catalyzed by poly(ADP-ribose) polymerases (PARPs) at DNA lesions, which facilitates DNA damage repair. Recent studies suggest that poly(ADP-ribosyl)ation is one of the first steps of cellular DNA damage response and governs early DNA damage response pathways. Suppression of DNA damage-induced poly(ADP-ribosyl)ation by PARP inhibitors impairs early DNA damage response events. Moreover, PARP inhibitors are emerging as anti-cancer drugs in phase III clinical trials for BRCA-deficient tumors. In this review, we discuss recent findings on poly(ADP-ribosyl)ation in DNA damage response as well as the molecular mechanism by which PARP inhibitors selectively kill tumor cells with BRCA mutations.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos

Recent studies have shown that DNA damage affects embryo development and also somatic cell reprogramming into induced pluripotent stem (iPS) cells. It has been also shown that treatment with histone deacetylase inhibitors (HDACi) improves development of embryos produced by somatic cell nuclear transfer (SCNT) and enhances somatic cell reprogramming. There is evidence that increasing histone acetylation at the sites of DNA double-strand breaks (DSBs) is critical for DNA damage repair. Therefore, we hypothesized that HDACi treatment enhances cell programming and embryo development by facilitating DNA damage repair. To test this hypothesis, we first established a DNA damage model wherein exposure of nuclear donor cells to ultraviolet (UV) light prior to nuclear transfer reduced the development of SCNT embryos proportional to the length of UV exposure. Detection of phosphorylated histone H2A.x (H2AX139ph) foci confirmed that exposure of nuclear donor cells to UV light for 10 s was sufficient to increase DSBs in SCNT embryos. Treatment with HDACi during embryo culture increased development and reduced DSBs in SCNT embryos produced from UV-treated cells. Transcript abundance of genes involved in either the homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways for DSBs repair was reduced by HDACi treatment in developing embryos at day 5 after SCNT. Interestingly, expression of HR and NHEJ genes was similar between HDACi-treated and control SCNT embryos that developed to the blastocyst stage. This suggested that the increased number of embryos that could achieve the blastocyst stage in response to HDACi treatment have repaired DNA damage. These results demonstrate that DNA damage in nuclear donor cells is an important component affecting development of SCNT embryos, and that HDACi treatment after nuclear transfer enhances DSBs repair and development of SCNT embryos.

RNF168-mediated H2A neddylation antagonizes its ubiquitination and regulates DNA damage repair

NEDD8 is an important regulatory factor in many biological processes. However, the substrates for neddylation, and the relationship between the ubiquitin and NEDD8 pathways remain largely unknown. Here, we show that NEDD8 is covalently conjugated to histone 2A (H2A), and that neddylation of H2A antagonizes its ubiquitylation. NEDD8 suppresses ubiquitylation of H2A, and a decreased level of free NEDD8 promotes H2A ubiquitylation. Furthermore, we found that the E3 ligase RNF168 promotes both H2A ubiquitylation and neddylation. Interestingly, RNF168 is itself a substrate for NEDD8, and neddylation of RNF168 is necessary for its E3 ubiquitin activity. Inhibition of RNF168 neddylation impairs the interaction between RNF168 and its E2 enzyme Ubc13 (also known as UBE2N). Moreover, in response to DNA damage, the level of H2A neddylation decreased with an increase in the ubiquitylation of H2A, which facilitates DNA damage repair. During the later stages of damage repair, H2A neddylation increased gradually, whereas ubiquitylation decreased to basal levels. Mechanistically, NEDD8 negatively regulates the DNA damage repair process through suppression of the ubiquitylation of H2A and γH2AX, which further blocks the recruitment of the damage response protein BRCA1. Our findings elucidate the relationship of H2A ubiquitylation and neddylation, and suggest a novel modulatory approach to DNA damage repair through the neddylation pathway.

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.

A Single Cohesin Complex Performs Mitotic and Meiotic Functions in the Protist Tetrahymena

The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.

BRCA2 Protein Deficiency Exaggerates Doxorubicin-induced Cardiomyocyte Apoptosis and Cardiac Failure

The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.

ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

Deoxynucleoside Salvage Facilitates DNA Repair During Ribonucleotide Reductase Blockade in Human Cervical Cancers

Cells generate 2'-deoxyribonucleoside triphosphates (dNTPs) for both replication and repair of damaged DNA predominantly through de novo reduction of intracellular ribonucleotides by ribonucleotide reductase (RNR). Cells can also salvage deoxynucleosides by deoxycytidine kinase/thymidine kinase 1 in the cytosol or by deoxyguanosine kinase/thymidine kinase 2 in mitochondria. In this study we investigated whether the salvage dNTP supply pathway facilitates DNA damage repair, promoting cell survival, when pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC no.663249) impairs the de novo pathway. Human cervical cancer cells were subjected to radiation with or without 3-AP under medium deoxynucleoside concentrations of 0, 0.05, 0.5 and 5.0µM. Efficacy of DNA damage repair was assessed by γ-H2AX flow cytometry and focus counts, by single cell electrophoresis (Comet assay), and by caspase 3 cleavage assay as a marker of treatment-induced apoptosis. Cell survival was assessed by colony formation. We found that deoxyribonucleotide salvage facilitates DNA repair during RNR inhibition by 3-AP and that salvage reduces the radiochemosensitivity of human cervical cancer cells.