Ismail, I. H., A. Dronyk, X. Hu, M. J. Hendzel, and A. R. Shaw, "BCL10 is recruited to sites of DNA damage to facilitate DNA double-strand break repair.", Cell cycle (Georgetown, Tex.), vol. 15, issue 1, pp. 84-94, 2016 Jan 2. Abstract

Recent studies have found BCL10 can localize to the nucleus and that this is linked to tumor aggression and poorer prognosis. These studies suggest that BCL10 localization plays a novel role in the nucleus that may contribute to cellular transformation and carcinogenesis. In this study, we show that BCL10 functions as part of the DNA damage response (DDR). We found that BCL10 facilitates the rapid recruitment of RPA, BRCA1 and RAD51 to sites of DNA damage. Furthermore, we also found that ATM phosphorylates BCL10 in response to DNA damage. Functionally, BCL10 promoted DNA double-strand breaks repair, enhancing cell survival after DNA damage. Taken together our results suggest a novel role for BCL10 in the repair of DNA lesions.

Ismail, I. H., J. - P. Gagné, M. - M. Genois, H. Strickfaden, D. McDonald, Z. Xu, G. G. Poirier, J. - Y. Masson, and M. J. Hendzel, "The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice.", Nature cell biology, vol. 17, issue 11, pp. 1446-57, 2015 Nov. Abstract

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

Ismail, I. H., R. Davidson, J. - P. Gagne, Z. Xu, G. Poirier, and M. J. Hendzel, "Germ-line Mutations in BAP1 Impair its Function in DNA Double-Strand break Repair.", Cancer research, 2014 Jun 3. Abstract

The BRCA1-associated deubiquitylase BAP1 is mutated in several cancers, most notably mesothelioma and melanoma, where it is thought to promote oncogenesis. In this study, we present evidence that BAP1 functions as part of the DNA damage response (DDR). We found that BAP1 mediates rapid poly(ADP-ribose)-dependent recruitment of the Polycomb deubiquitylase complex PR-DUB to sites of DNA damage. Further, we identified BAP1 as a phosphorylation target for the DDR kinase ATM. Functionally, BAP1 promoted repair of DNA double-strand breaks, enhancing cell survival after DNA damage. Our results highlight the importance of ubiquitin turnover at sites of DNA damage, and they provide a mechanism to account for the tumor suppressor function of BAP1.

Gieni, R. S., I. H. Ismail, S. Campbell, and M. J. Hendzel, "Polycomb group proteins in the DNA damage response: a link between radiation resistance and "stemness".", Cell Cycle, vol. 10, no. 6, United States, pp. 883-94, 3, 2011. Abstract

Polycomb group proteins, which have well-established roles in gene regulation, were recently found to accumulate on chromatin surrounding DNA damage and to contribute up to 40 percent of the radiation resistance of cell lines. The oncogenic polycomb protein, BMI-1, was additionally shown to be essential for the increased radiation resistance observed in stem cells and cancer stem cells relative to their more differentiated counterparts. BMI-1, is a very early DNA damage response protein that accumulates through a $\gamma$H2AX/RNF8-independent, but poly(ADP-ribosyl)ation-dependent mechanism at DNA double-strand breaks. BMI-1 acts together with RING2 and other components of the PRC1 histone H2A E3 ubiquitin ligase to ubiquitylate histones H2A and H2AX in response to DNA damage. BMI-1 dependent ubiquitin modifications are at the base of an ubiquitin pathway that enhances radioresistance through the accumulation of RAP80, 53BP1, and BRCA1. Members of the PRC2 histone H3 lysine 27 methyltransferase complex are also recruited to sites of DSBs but it remains to be determined whether the histone methyltransferase and histone E3 ubiquitin ligase polycomb complexes function in concert or independently during DNA repair. Understanding the contribution of polycomb group proteins to the DNA damage response may lead to novel therapeutic strategies that increase the response of human cancers to therapies that work through DNA damage, while simultaneously sensitizing the cancer stem cell population that would otherwise lead to relapse

Campbell, S., I. H. Ismail, L. C. Young, G. G. Poirier, and M. J. Hendzel, "Polycomb repressive complex 2 contributes to DNA double-strand break repair.", Cell Cycle, vol. 12, no. 16, United States, pp. 2675-83, 8, 2013. Abstract

Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy

Ismail, I. H., and M. J. Hendzel, "The gamma-H2A.X: is it just a surrogate marker of double-strand breaks or much more?", Environ Mol Mutagen, vol. 49, no. 1, United States, pp. 73-82, 1, 2008. Abstract

In recent years, several histone modifications have been implicated in the cellular response to DNA double-strand breaks (DSBs). One of the best characterized histone modifications important in DSB repair is the phosphorylation of histone H2A variant, H2A.X. In response to DSBs, H2A.X is phosphorylated and this phosphorylation is required for DSB signaling and the retention of repair proteins at the break site. Despite the existing picture that the function of H2A.X is to promote DNA repair, very recent data suggest that the phosphorylation of histone H2A.X has additional functions. This is analogous to histone H3 phosphorylation on serine 10, which participates in seemingly incompatible functions–transcriptional activation and mitosis. In this review, we discuss the role of histone H2A.X in maintaining genomic stability and review emerging evidence that histone H2A.X is multifunctional

Elmroth, K., J. Nygren, S. M$\aa$rtensson, I. H. Ismail, and O. Hammarsten, "Cleavage of cellular DNA by calicheamicin gamma1.", DNA Repair (Amst), vol. 2, no. 4, Netherlands, pp. 363-74, 4, 2003. Abstract

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0 degrees C as compared to 37 degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calicheamicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease

Ismail, I. H., S. Nyström, J. Nygren, and O. Hammarsten, "Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks.", J Biol Chem, vol. 280, no. 6, United States, pp. 4649-55, 2, 2005. Abstract

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells

Ismail, I. H., D. McDonald, H. Strickfaden, Z. Xu, and M. J. Hendzel, "A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks.", J Biol Chem, vol. 288, no. 37, United States, pp. 26944-54, 9, 2013. Abstract

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks

Ismail, I. H., C. Andrin, D. McDonald, and M. J. Hendzel, "BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair.", J Cell Biol, vol. 191, no. 1, United States, pp. 45-60, 10, 2010. Abstract

Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of $\gamma$-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability

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