Watching the DNA Repair Ensemble Dance

AbstractRepair of damaged DNA is a dynamic process that requires careful orchestration of a multitude of enzymes, adaptor proteins, and chromatin constituents. In this issue of Cell, Lisby et al. (2004) provide a visual glimpse into how the diverse signaling and repair machines are organized in space and time around the deadliest genetic lesions—the DNA double-strand breaks

Elucidating the role of DNA damage and human cytomegalovirus in medulloblastoma and glioblastoma

The most common primary malignant brain tumor in children is Medulloblastoma, while Glioblastoma is the most common in adults. Treatment for both include some combination of surgery, radiation therapy, and chemotherapy. The evolution of most primary malignant brain tumors is unknown, although varying degree of genomic instability caused by defects in the DNA Damage Response (DDR) is suspected. Lately, even human cytomegalovirus (HCMV) has emerged as a suspected pathogen possibly implicated in malignant tumor evolution. Nevertheless, the causes of the chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies (i.e. radiation and chemotherapy) remain largely unknown. Thus, the main aim of this PhD thesis is to investigate the role of HCMV in the context of DDR in human Medulloblastoma and Glioblastoma. In the 1st study, we turned our attention towards Glioblastoma (GBM). We examined the ability of HCMV to induce a more aggressive cancer stem cell (CSC)-like phenotype in primary GBM cell lines. HCMV infection induced a stem cell phenotype in primary GBM cell lines as determined by changes in the cellular gene expression profile and by the conferred ability of cells to grow as neurospheres in vitro, and this phenotype was prevented by treatment with the anti-viral drug ganciclovir. As CSCs are known to be resistant to chemotherapy, our results imply that HCMV may enhance the malignancy grade of the tumor, and possibly contribute to therapy resistance. In the 2nd study, we found pronounced endogenous DNA damage signaling and constitutive activation of DNA damage checkpoint kinase cascades across our medulloblastoma cohort. The bulk of the specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection. Our results strongly indicate that in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair mechanisms. In the 3rd study, we examined cancer stem cell markers (CD133, CD15, VEGFR2) and HCMV protein expression in human medulloblastoma specimens and medulloblastoma cell lines, at the same time considering also the replication stress and DNA damage response, as cancer stem cells are often more resistant to standard-of-care radiation and chemotherapy treatments. Our immunohistochemistry analysis on clinical material identified widespread expression of the VEGFR2 receptor and CD15, yet more limited expression of CD133 compared to GBM. In addition, assessments of expression of HCMV early and late proteins have been carried out in parallel, along with cell culture experiments with HCMV infection and replication stress responses in medulloblastoma cell lines. Remarkably, we found that unlike the ‘non-stem cell’ medulloblastoma cell lines, the cell line that showed robust stemness phenotype featured a very distinct response to DNA replication stress and HCMV infection, both emerging hallmarks of brain cancers. In the 4th study, we show that HCMV infection induced replication stress (RS) and triggered host DNA damage response (DDR) in permissive and non-permissive human cells. Further, we show that undergoing standard-of-care genotoxic radiochemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV markers after tumor recurrence. We propose a model to explain oncomodulatory effects of HCMV, through RS induction, DDR subversion, cell death inhibition and host-cell’s genome destabilization. Our findings provide fresh insights into HCMV pathobiology and inspiration for future strategies to combine radio-chemotherapy with anti-viral drugs for cancer treatment

SUMO Boosts the DNA Damage Response Barrier against Cancer

Cells exposed to genotoxic insults such as ionizing radiation activate a signaling cascade to repair the damaged DNA. Two recent articles published in Nature show that such genome maintenance requires modifications of tumor suppressor proteins BRCA1 and 53BP1 by the small ubiquitin-like modifier SUMO

Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1

53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint

Expression of a p16INK4a-specific ribozyme downmodulates p16INK4a abundance and accelerates cell proliferation

AbstractThe p16INK4a tumor suppressor negatively regulates progression through the G1 phase of the mammalian cell cycle. To mimic the downmodulation of p16INK4a commonly seen in cancer, we designed and characterized a hammerhead ribozyme against exon E1α of the murine p16INK4a transcript. Stable expression of the ribozyme in murine erythroleukemia (MEL) cells reduced the endogenous p16INK4a protein by more than 70% and significantly accelerated cell cycle progression. The specificity and efficiency of our new ribozyme suggest its possible application in elucidating the role of p16INK4a in fundamental biological processes including homeostatic tissue renewal, protection against oncogenic transformation, and cellular senescence

USP7 counteracts SCFβTrCP- but not APCCdh1-mediated proteolysis of Claspin

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCFβTrCP-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase–specific, SCFβTrCP-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCFβTrCP- but not APCCdh1-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR–Chk1 pathway

Regulation of Replication Fork Progression Through Histone Supply and Demand

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand

Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN–MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1–NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage