Publications by authors named "Raj K Pandita"

56 Publications

Role of HP1β during spermatogenesis and DNA replication.

Chromosoma 2020 12 10;129(3-4):215-226. Epub 2020 Jul 10.

Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX, 77030, USA.

Heterochromatin protein 1β (HP1β), encoded by the Cbx1 gene, has been functionally linked to chromatin condensation, transcriptional regulation, and DNA damage repair. Here we report that testis-specific Cbx1 conditional knockout (Cbx1 cKO) impairs male germ cell development in mice. Depletion of HP1β negatively affected sperm maturation and increased seminiferous tubule degeneration in Cbx1 cKO mice. In addition, the spermatogonia have elevated γ-H2AX foci levels as do Cbx1 deficient mouse embryonic fibroblasts (MEFs) as compared to wild-type (WT) control MEFs. The increase in γ-H2AX foci in proliferating Cbx1 cKO cells indicates defective replication-dependent DNA damage repair. Depletion or loss of HP1β from human cells and MEFs increased DNA replication fork stalling and firing of new origins of replication, indicating defective DNA synthesis. Taken together, these results suggest that loss of HP1β in proliferating cells leads to DNA replication defects with associated DNA damage that impact spermatogenesis.
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http://dx.doi.org/10.1007/s00412-020-00739-4DOI Listing
December 2020

Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.

Commun Biol 2019 5;2:253. Epub 2019 Jul 5.

1Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030 USA.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.
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http://dx.doi.org/10.1038/s42003-019-0498-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611875PMC
May 2020

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Hepatology 2019 06 13;69(6):2608-2622. Epub 2019 Mar 13.

Department of Cancer and Cell Biology, University of Cincinnati, Cincinnati, OH.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.
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http://dx.doi.org/10.1002/hep.30529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541504PMC
June 2019

Complete Local and Abscopal Responses from a Combination of Radiation and Nivolumab in Refractory Hodgkin's Lymphoma.

Radiat Res 2018 09 27;190(3):322-329. Epub 2018 Jun 27.

Department of a   Internal Medicine, Houston Methodist Research Institute, Houston, Texas 77030.

Until recently, patients with relapsed Hodgkin's lymphoma after brentuximab vedotin (Bv) treatments had poor treatment outcomes. Checkpoint inhibitors such as nivolumab and pembrolizumab that bind to and inhibit programmed cell death protein-1 (PD-1), have demonstrated an overall response rate of 70% in Hodgkin's lymphoma patients; however, complete response is still low at 20% with median progression-free survival of 14 months. There are ongoing clinical studies to seek out synergistic combinations, with the goal of improving the complete response rates for the cure of Hodgkin's lymphoma. Although radiotherapy has a limited survival benefit in such refractory patients, several preclinical models and anecdotal clinical evidence have suggested that combining local tumor irradiation with checkpoint inhibitors can produce systemic regression of distant tumors, an abscopal effect. Most of these reported studies on the response with local conformal radiotherapy and checkpoint inhibitors in combination with the anti-cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) antibody-ipilimumab are in melanoma. Here we report in our case series that the checkpoint inhibitors that block CTLA4 and B7-homolog 1 (B7-H1) or PD-1 in preclinical radiotherapy models have shown an increased the rate of tumor regression. Our case series demonstrates that combining local irradiation with anti-PD-1 checkpoint blockade treatment is feasible and synergistic in refractory Hodgkin's lymphoma. Correlative studies also suggest that the expression of programmed death-ligand 1 (PD-L1), DNA damage response and mutational tumor burden can be used as potential biomarkers for treatment response.
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http://dx.doi.org/10.1667/RR15048.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135239PMC
September 2018

SMARCAD1 Phosphorylation and Ubiquitination Are Required for Resection during DNA Double-Strand Break Repair.

iScience 2018 04;2:123-135

Department of Radiation Oncology, Houston Methodist Cancer Center, The Houston Methodist Research Institute, Weill Cornell Medical College, Houston, TX 77030, USA. Electronic address:

The chromatin remodeling factor SMARCAD1, an SWI/SNF ATPase family member, has a role in 5' end resection at DNA double-strand breaks (DSBs) to produce single-strand DNA (ssDNA), a critical step for subsequent checkpoint and repair factor loading to remove DNA damage. However, the mechanistic details of SMARCAD1 coupling to the DNA damage response and repair pathways remains unknown. Here we report that SMARCAD1 is recruited to DNA DSBs through an ATM-dependent process. Depletion of SMARCAD1 reduces ionizing radiation (IR)-induced repairosome foci formation and DSB repair by homologous recombination (HR). IR induces SMARCAD1 phosphorylation at a conserved T906 by ATM kinase, a modification essential for SMARCAD1 recruitment to DSBs. Interestingly, T906 phosphorylation is also important for SMARCAD1 ubiquitination by RING1 at K905. Both these post-translational modifications are critical for regulating the role of SMARCAD1 in DNA end resection, HR-mediated repair, and cell survival after DNA damage.
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http://dx.doi.org/10.1016/j.isci.2018.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993204PMC
April 2018

Harnessing and Optimizing the Interplay between Immunotherapy and Radiotherapy to Improve Survival Outcomes.

Mol Cancer Res 2018 08 28;16(8):1209-1214. Epub 2018 Mar 28.

Department of Radiation Oncology, the Houston Methodist Research Institute, Weil Cornell Medical College, Houston, Texas.

In the past, radiotherapy was primarily used to control local disease, but recent technological advances in accurate, high-dose ionizing radiation (IR) delivery have not only increased local tumor control but in some cases reduced metastatic burden. These "off target" therapeutic effects of IR at nonirradiated tumor sites, also known as abscopal effects, are thought to be mediated by tumor antigen-primed T cells that travel to metastatic sites and promote tumor regression. Similarly, early indications reveal that IR in combination with immune checkpoint inhibitors, such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1), can provide superior therapeutic responses. These observations suggest that local radiotherapy results in altered gene expression, exposure of new antigens, or cell death that can interact with immunotherapy. As such, radiotherapy enhancement of immune responses offers a promising synergy with the potential for substantial clinical benefit. This review focuses on the biology that underlies the mechanisms for the interaction between radiation-induced tumor cell death and enhanced immunologic response. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072560PMC
August 2018

β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination.

Mol Cell Biol 2018 05 16;38(9). Epub 2018 Apr 16.

Department of Radiation Oncology, Weill Cornell Medical College, The Houston Methodist Research Institute, Houston, Texas, USA

The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated β1-integrin is associated with enhanced breast cancer cell survival postirradiation, but how β1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA double-strand break (DSB) repair, and we found that nonmalignant breast epithelial (S1) cells with low β1-integrin expression have a higher frequency of S-phase-specific IR-induced chromosomal aberrations than the derivative malignant breast (T4-2) cells with high β1-integrin expression. In addition, there was an increased frequency of IR-induced homologous recombination (HR) repairosome focus formation in T4-2 cells compared with that of S1 cells. Cellular levels of Rad51 in T4-2 cells, a critical factor in HR-mediated DSB repair, were significantly higher. Blocking or depleting β1-integrin activity in T4-2 cells reduced Rad51 levels, while ectopic expression of β1-integrin in S1 cells correspondingly increased Rad51 levels, suggesting that Rad51 is regulated by β1-integrin. The low level of Rad51 protein in S1 cells was found to be due to rapid degradation by the ubiquitin proteasome pathway (UPP). Furthermore, the E3 ubiquitin ligase RING1 was highly upregulated in S1 cells compared to T4-2 cells. Ectopic β1-integrin expression in S1 cells reduced RING1 levels and increased Rad51 accumulation. In contrast, β1-integrin depletion in T4-2 cells significantly increased RING1 protein levels and potentiated Rad51 ubiquitination. These data suggest for the first time that elevated levels of the extracellular matrix receptor β1-integrin can increase tumor cell radioresistance by decreasing Rad51 degradation through a RING1-mediated proteasomal pathway.
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http://dx.doi.org/10.1128/MCB.00672-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902589PMC
May 2018

MOF Suppresses Replication Stress and Contributes to Resolution of Stalled Replication Forks.

Mol Cell Biol 2018 03 27;38(6). Epub 2018 Feb 27.

Department of Radiation Oncology, Weill Cornell Medical College, The Houston Methodist Research Institute, Houston, Texas, USA

The human MOF (hMOF) protein belongs to the MYST family of histone acetyltransferases and plays a critical role in transcription and the DNA damage response. MOF is essential for cell proliferation; however, its role during replication and replicative stress is unknown. Here we demonstrate that cells depleted of MOF and under replicative stress induced by cisplatin, hydroxyurea, or camptothecin have reduced survival, a higher frequency of S-phase-specific chromosome damage, and increased R-loop formation. MOF depletion decreased replication fork speed and, when combined with replicative stress, also increased stalled replication forks as well as new origin firing. MOF interacted with PCNA, a key coordinator of replication and repair machinery at replication forks, and affected its ubiquitination and recruitment to the DNA damage site. Depletion of MOF, therefore, compromised the DNA damage repair response as evidenced by decreased Mre11, RPA70, Rad51, and PCNA focus formation, reduced DNA end resection, and decreased CHK1 phosphorylation in cells after exposure to hydroxyurea or cisplatin. These results support the argument that MOF plays an important role in suppressing replication stress induced by genotoxic agents at several stages during the DNA damage response.
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http://dx.doi.org/10.1128/MCB.00484-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829482PMC
March 2018

Deletion in Head and Neck Cancer Cells Is Associated with CDK2 Activation, Replication Stress, and Vulnerability to CHK1 Inhibition.

Cancer Res 2018 02 11;78(3):781-797. Epub 2017 Dec 11.

Department of Head and Neck Surgery, University of Texas MD Anderson Cancer Center, Houston, Texas.

Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC value greater than the cell panel median and lacked early S-phase arrest. Novel association between copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811346PMC
February 2018

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination.

Stem Cell Reports 2017 11 2;9(5):1660-1674. Epub 2017 Nov 2.

Department of Radiation Oncology, Weill Cornell Medical College, The Houston Methodist Hospital Research Institute, Houston, TX 77030, USA. Electronic address:

The nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on double-strand break (DSB) repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831054PMC
November 2017

Erratum: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin.

Sci Rep 2017 10 10;7(1):12932. Epub 2017 Oct 10.

Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State, 500007, India.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-017-12314-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635132PMC
October 2017

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.

Sci Rep 2017 09 14;7(1):11541. Epub 2017 Sep 14.

Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi, 110025, India.

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.
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http://dx.doi.org/10.1038/s41598-017-11177-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599563PMC
September 2017

miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin.

Sci Rep 2017 06 27;7(1):4263. Epub 2017 Jun 27.

Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana State, 500007, India.

The B-lymphoma Moloney murine leukemia virus insertion region-1 protein (BMI1) acts as an oncogene in various cancers, including breast cancer. Recent evidence suggests that BMI1 is rapidly recruited to sites of DNA double strand breaks where it facilitates histone H2A ubiquitination and DNA double strand break repair by homologous recombination. Here we show that miR-15a and miR-16 expression is decreased during the initial period after DNA damage where it would otherwise down-regulate BMI1, impairing DNA repair. Elevated miR-15a and miR-16 levels down-regulated BMI1 and other polycomb group proteins like RING1A, RING1B, EZH2 and also altered the expression of proteins associated with the BMI1 dependent ubiquitination pathway. Antagonizing the expression of miR-15a and miR-16, enhanced BMI1 protein levels and increased DNA repair. Further, overexpression of miR-15a and miR-16 sensitized breast cancer cells to DNA damage induced by the chemotherapeutic drug doxorubicin. Our results suggest that miR-15a and miR-16 mediate the down-regulation of BMI1, which impedes DNA repair while elevated levels can sensitize breast cancer cells to doxorubicin leading to apoptotic cell death. This data identifies a new target for manipulating DNA damage response that could impact the development of improved therapeutics for breast cancer.
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http://dx.doi.org/10.1038/s41598-017-02800-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487337PMC
June 2017

Aurora kinase B dependent phosphorylation of 53BP1 is required for resolving merotelic kinetochore-microtubule attachment errors during mitosis.

Oncotarget 2017 Jul;8(30):48671-48687

Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA.

Defects in resolving kinetochore-microtubule attachment mistakes during mitosis is linked to chromosome instability associated with carcinogenesis as well as resistance to cancer therapy. Here we report for the first time that tumor suppressor p53-binding protein 1 (53BP1) is phosphorylated at serine 1342 (S1342) by Aurora kinase B both in vitro and in human cells, which is required for optimal recruitment of 53BP1 at kinetochores. Furthermore, 53BP1 staining normally localized on the outer kinetochore, extended to the whole kinetochore when it is merotelically-attached, in concert with mitotic centromere-associated kinesin. Kinetochore-binding of pS1342-53BP1 is essential for efficient resolving of merotelic attachment, a spontaneous kinetochore-microtubule connection error that usually causes aneuploidy. Consistently, loss of 53BP1 results in significant increase in lagging chromosome events, micronuclei formation and aneuploidy, due to the unresolved merotely in both cancer and primary cells, which is prevented by ectopic wild type 53BP1 but not by the nonphophorylable S1342A mutant. We thus document a novel DNA damage-independent function of 53BP1 in maintaining faithful chromosome segregation during mitosis.
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http://dx.doi.org/10.18632/oncotarget.16225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564716PMC
July 2017

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Blood 2017 05 7;129(18):2479-2492. Epub 2017 Mar 7.

QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both and were constitutively or conditionally deleted. Constitutive double knockout (DKO) caused early embryonic lethality, whereas conditional double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of or Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.
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http://dx.doi.org/10.1182/blood-2016-06-725093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418634PMC
May 2017

MCL-1 Depletion Impairs DNA Double-Strand Break Repair and Reinitiation of Stalled DNA Replication Forks.

Mol Cell Biol 2017 02 19;37(3). Epub 2017 Jan 19.

Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, Texas, USA

Myeloid cell leukemia 1 (MCL-1) is a prosurvival BCL-2 protein family member highly expressed in hematopoietic stem cells (HSCs) and regulated by growth factor signals that manifest antiapoptotic activity. Here we report that depletion of MCL-1 but not its isoform MCL-1S increases genomic instability and cell sensitivity to ionizing radiation (IR)-induced death. MCL-1 association with genomic DNA increased postirradiation, and the protein colocalized with 53BP1 foci. Postirradiation, MCL-1-depleted cells exhibited decreased γ-H2AX foci, decreased phosphorylation of ATR, and higher levels of residual 53BP1 and RIF1 foci, suggesting that DNA double-strand break (DSB) repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1-depleted cells had a reduced frequency of IR-induced BRCA1, RPA, and Rad51 focus formation, decreased DNA end resection, and decreased HR repair in the DR-GFP DSB repair model. Similarly, after HU induction of stalled replication forks in MCL-1-depleted cells, there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR-mediated resolution of collapsed forks. Therefore, the present data support a model whereby MCL-1 depletion increases 53BP1 and RIF1 colocalization at DSBs, which inhibits BRCA1 recruitment, and sensitizes cells to DSBs from IR or stalled replication forks that require HR for repair.
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http://dx.doi.org/10.1128/MCB.00535-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247612PMC
February 2017

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes.

Nat Commun 2016 10 5;7:13049. Epub 2016 Oct 5.

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555, USA.

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.
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http://dx.doi.org/10.1038/ncomms13049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059474PMC
October 2016

Pluripotent Stem Cells and DNA Damage Response to Ionizing Radiations.

Radiat Res 2016 07 22;186(1):17-26. Epub 2016 Jun 22.

Department of Radiation Oncology, The Houston Methodist Research Institute, Weill Cornell Medical College, The Houston Methodist Hospital, Houston, Texas 77030.

Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences.
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http://dx.doi.org/10.1667/RR14417.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963261PMC
July 2016

HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells.

Cancer Res 2016 08 14;76(15):4443-56. Epub 2016 Jun 14.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is overexpressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor-negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel, or carboplatin by suppressing apoptosis and upregulating NF-κB Overexpressed HOXC10 increases S-phase-specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double-strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin-dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer. Cancer Res; 76(15); 4443-56. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970943PMC
August 2016

β2-spectrin depletion impairs DNA damage repair.

Oncotarget 2016 Jun;7(23):33557-70

Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA.

β2-Spectrin (β2SP/SPTBN1, gene SPTBN1) is a key TGF-β/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-β signaling and can contribute to liver cancer development. Here we report that cells deficient in β2-Spectrin (β2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), β2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, β2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in β2SP deficient cells. We propose that β2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination.
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http://dx.doi.org/10.18632/oncotarget.9677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085102PMC
June 2016

The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation.

Mol Cell 2016 05;62(3):409-421

St. Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Quebec City, QC G1R 3S3, Canada; Centre Hospitalier Universitaire de Québec Research Center and School of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada. Electronic address:

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.
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http://dx.doi.org/10.1016/j.molcel.2016.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887106PMC
May 2016

Torin2 Suppresses Ionizing Radiation-Induced DNA Damage Repair.

Radiat Res 2016 05 2;185(5):527-38. Epub 2016 May 2.

a   Department of Radiation Oncology and.

Several classes of inhibitors of the mammalian target of rapamycin (mTOR) have been developed based on its central role in sensing growth factor and nutrient levels to regulate cellular metabolism. However, its ATP-binding site closely resembles other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, resulting in reactivity with these targets that may also be therapeutically useful. The ATP-competitive mTOR inhibitor, Torin2, shows biochemical activity against the DNA repair-associated proteins ATM, ATR and DNA-PK, which raises the possibility that Torin2 and related compounds might radiosensitize cancerous tumors. In this study Torin2 was also found to enhance ionizing radiation-induced cell killing in conditions where ATM was dispensable, confirming the requirement for multiple PIKK targets. Moreover, Torin2 did not influence the initial appearance of γ-H2AX foci after irradiation but significantly delayed the disappearance of radiation-induced γ-H2AX foci, indicating a DNA repair defect. Torin2 increased the number of radiation-induced S-phase specific chromosome aberrations and reduced the frequency of radiation-induced CtIP and Rad51 foci formation, suggesting that Torin2 works by blocking homologous recombination (HR)-mediated DNA repair resulting in an S-phase specific DNA repair defect. Accordingly, Torin2 reduced HR-mediated repair of I-Sce1-induced DNA damage and contributed to replication fork stalling. We conclude that radiosensitization of tumor cells by Torin2 is associated with disrupting ATR- and ATM-dependent DNA damage responses. Our findings support the concept of developing combination cancer therapies that incorporate ionizing radiation therapy and Torin2 or compounds with similar properties.
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http://dx.doi.org/10.1667/RR14373.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922265PMC
May 2016

ATM functions at the peroxisome to induce pexophagy in response to ROS.

Nat Cell Biol 2015 Oct 7;17(10):1259-1269. Epub 2015 Sep 7.

Center for Translational Cancer Research, Institute for Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX 77030, USA.

Peroxisomes are highly metabolic, autonomously replicating organelles that generate reactive oxygen species (ROS) as a by-product of fatty acid β-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to ROS, ATM signalling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser 141, which promotes PEX5 monoubiquitylation at Lys 209, and recognition of ubiquitylated PEX5 by the autophagy adaptor protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy.
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http://dx.doi.org/10.1038/ncb3230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589490PMC
October 2015

Role of the Exocyst Complex Component Sec6/8 in Genomic Stability.

Mol Cell Biol 2015 Nov 17;35(21):3633-45. Epub 2015 Aug 17.

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA The Houston Methodist Research Institute, Houston, Texas, USA

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.
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http://dx.doi.org/10.1128/MCB.00768-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589602PMC
November 2015

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation.

J Biol Chem 2015 Oct 5;290(41):24636-48. Epub 2015 Aug 5.

From the Department of Internal Medicine, Sealy Center for Molecular Medicine,

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.
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http://dx.doi.org/10.1074/jbc.M115.658146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598976PMC
October 2015

Targeted inhibition of histone deacetylases and hedgehog signaling suppress tumor growth and homologous recombination in aerodigestive cancers.

Am J Cancer Res 2015 15;5(4):1337-52. Epub 2015 Mar 15.

Division of Molecular Radiation Biology, Department of Radiation Oncology, Harold C. Simmons Comprehensive Cancer Center, University of Texas at Southwestern Medical Center Dallas, TX, USA ; Anchorage and Valley Radiation Therapy Center Anchorage, AK, USA.

Standard combined modality therapies for aerodigestive tract malignancies have suboptimal outcomes, and targeting cancer-specific molecular pathways in combination with radiation could improve the therapeutic ratio. Dysregulation of epigenetic modulators such as histone deacetylases (HDACs), and developmental morphogens such as the hedgehog (HH) pathway have been implicated in aerodigestive tumor progression and metastasis. We hypothesized that simultaneous targeting of HDACs and the HH-pathway mediator Smoothened (Smo) represents an opportunity to overcome therapeutic resistance in these cancers. We evaluated the effects of the HDAC inhibitor SAHA and Smo inhibitor GDC-0449 with radiation in multiple aerodigestive cancer cell lines. Isobologram analyses showed that SAHA and GDC-0449 synergistically suppressed cancer cell proliferation in vitro. SAHA and GDC-0449 cooperatively enhanced G0/G1 cell cycle arrest which was associated with up-regulation of p21(waf). GDC-0449 prevented SAHA-induced up-regulation of Gli-1 and Gli-2. Both Smo and Ptc-1 expression was cooperatively suppressed by SAHA and GDC-0449. The combination of SAHA and GDC-0449 induced radiation sensitization with 2 Gy as determined by colony formation assays and cytogenetic analyses, which correlated with higher residual γ-H2AX and 53BP1 foci. In mouse tumor xenografts of the SqCC/Y1 cell line, SAHA and GDC-0449 delayed tumor growth longer and prolonged survival more than either agent alone. In summary, we have identified synergistic effect of HDAC and HH signaling for radiosensitization to improve therapeutic outcomes for aerodigestive malignancies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473314PMC
June 2015

BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1.

Proc Natl Acad Sci U S A 2015 Mar 2;112(11):E1210-9. Epub 2015 Mar 2.

Department of Cancer and Cell Biology, College of Medicine, University of Cincinnati, Cincinnati, OH 45267;

The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.
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http://dx.doi.org/10.1073/pnas.1418335112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371995PMC
March 2015

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

PLoS Genet 2015 Jan 29;11(1):e1004749. Epub 2015 Jan 29.

Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, United States of America.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.
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http://dx.doi.org/10.1371/journal.pgen.1004749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310589PMC
January 2015

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

Cancer Res 2015 Mar 14;75(5):858-69. Epub 2015 Jan 14.

Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, Texas. Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-2289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351820PMC
March 2015

MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

Cell Rep 2014 Jul 19;8(1):177-89. Epub 2014 Jun 19.

Deparment of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030, USA. Electronic address:

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.
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http://dx.doi.org/10.1016/j.celrep.2014.05.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300955PMC
July 2014