MDC1 collaborates with TopBP1 in DNA replication checkpoint control

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1-MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.     

The in vivo dynamic interplay of MDC1 and 53BP1 at DNA damage-induced nuclear foci

MDC1 (NFBD1) and 53BP1 are critical mediators of the mammalian DNA damage response (DDR) at nuclear foci. Here we show by quantitative imaging assays that MDC1 and 53BP1 are similar in total copy number (~1200 copies per focus), but differ substantially in dynamics at both replication-associated nuclear bodies in normal cells and DNA repair foci in ionizing radiation (IR)-damaged cells. The majority of MDC1 (~80%) is extremely mobile and under continuous exchange, with only a small fraction (~20%) remaining immobile at foci irrespective of IR treatment. By contrast, 53BP1 has a smaller mobile fraction (~35%) and a larger immobile fraction (~65%) at nuclear bodies, and becomes more dynamic (~20% increase in mobile pool) upon IR-induced DNA damage. More specifically, the dynamics of 53BP1 is dependent on a minimal foci-targeting region (1231-1709), and differentially regulated by its N-terminus (1-1231) and C-terminal tBRCT domain (1709-1972). Furthermore, MDC1 knockdown, or disruption of 53BP1-MDC1 interaction, reduced the number of 53BP1 molecules at foci by ~60%, but only modestly affected 53BP1 retention. This novel in vivo evidence reveals distinct dynamics of MDC1 and 53BP1 at different types of nuclear structures, and shows that MDC1 directly recruits and retains a subset of 53BP1 for DNA repair.     

MDC1 regulates DNA-PK autophosphorylation in response to DNA damage

DNA damage initiates signaling events through kinase cascades that result in cell cycle checkpoint control and DNA repair. However, it is not yet clear how the signaling pathways relay to DNA damage repair. Using the repeat region of checkpoint protein MDC1 (mediator of DNA damage checkpoint protein 1), we identified DNA-PKcs/Ku as MDC1-associated proteins. Here, we show that MDC1 directly interacts with the Ku/DNA-PKcs complex. Down-regulation of MDC1 resulted in defective phospho-DNA-PKcs foci formation and DNA-PKcs autophosphorylation, suggesting that MDC1 regulates autophosphorylation of DNA-PKcs following DNA damage. Furthermore, DNA-PK-dependent DNA damage repair is defective in cells depleted of MDC1. Taken together, these results suggest that the MDC1 repeat region is involved in protein-protein interaction with DNA-PKcs/Ku, and MDC1 regulates DNA damage repair by influencing DNA-PK autophosphorylation. Therefore, MDC1 acts not only as a mediator of DNA damage checkpoint but also as a mediator of DNA damage repair.     

MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes

The protein MDC1/NFBD1 contains a forkhead-associated (FHA) domain and two BRCA1 carboxyl-terminal (BRCT) domains. It interacts with several proteins involved in DNA damage repair and checkpoint signalling, and is phosphorylated in response to DNA damage and during mitosis. Upon treatment of cultured human cells with DNA damaging agents, MDC1/NFBD1 translocates to sites of DNA lesions, where it collaborates with other proteins and with phosphorylated histone H2AX to mediate the accumulation of checkpoint and repair factors into nuclear foci. Down-regulation of MDC1/NFBD1 expression levels by small interfering RNA (siRNA) renders cells hyper-sensitive to DNA damaging agents and leads to defects in cell cycle checkpoint activation and apoptosis. Thus, MDC1/NFBD1 appears to be a key regulator of the DNA damage response in mammalian cells.     

MDC1 maintains active elongation complexes of RNA polymerase II

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Recruitment of proteins to DNA double-strand breaks: MDC1 directly recruits RAP80

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. Occurrence of DSBs in the cell activates the DNA damage response (DDR), which involves signaling cascades that sense and respond to the damage. Promptly after DSB induction, DDR proteins accumulate surrounding both DNA ends and form microscopically-visible foci. Recently, we demonstrated that the key DDR protein MDC1 directly binds RAP80, an additional DDR protein that recruits BRCA1 to DSBs. We provided evidences that the MDC1-RAP80 interaction depends on a ubiquitylation event on K-1977 of MDC1. However, it remained unknown whether K-1977 of MDC1 is required for the recruitment of RAP80 to DSBs. Here we show that K-1977 of MDC1 is necessary for focus formation by RAP80. Nevertheless, it has not effect on focus formation by γ-H2AX, MDC1 or 53BP1. The results imply a role for the MDC1-RAP80 interaction in focus formation by the RAP80-BRCA1 complex. In light of these recent results we discuss several aspects of the complexity of focus formation and present a model for the involvement of individual and complex recruitment mechanisms in focus formation.     

Disassembly of MDC1 foci is controlled by ubiquitin-proteasome-dependent degradation

The orderly recruitment, retention, and disassembly of DNA damage response proteins at sites of damaged DNA is a conserved process throughout eukaryotic evolution. The recruitment and retention of DNA repair factors in foci is mediated by a complex network of protein-protein interactions; however, the mechanisms of focus disassembly remain to be defined. Mediator of DNA damage checkpoint protein 1 (MDC1) is an early and key component of the genome surveillance network activated by DNA double-strand breaks (DSBs). Here, we investigated the disassembly of MDC1 foci. First, we show that ubiquitylation directs the MDC1 protein for proteasome-dependent degradation. Ubiquitylated MDC1 associates with chromatin before and after exposure of cells to ionizing radiation (IR). In addition, increased MDC1 ubiquitylation in the chromatin fraction is observed in response to IR, which is correlated with a reduction in total MDC1 protein levels. We demonstrate that blocking MDC1 degradation by proteasome inhibitors leads to a persistence of MDC1 foci. Consistent with this observation, chromatin immunoprecipitation experiments reveal increased MDC1 protein at site-specific DSBs. Interestingly, we show that the persistence of MDC1 foci is associated with an abrogated recruitment of the downstream factor BRCA1 in a manner that is RNF8 independent. Collectively, the evidence presented here supports a novel mechanism for the disassembly of MDC1 foci via ubiquitin-proteasome dependent degradation, which appears to be a key step for the efficient assembly of BRCA1 foci.     

Mediator of DNA Damage Checkpoint 1 (MDC1) Regulates Mitotic Progression

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053–32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.     

Structural Insights into Recognition of MDC1 by TopBP1 in DNA Replication Checkpoint Control

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NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer

A large nuclear protein of 2089 amino acids, NFBD1/MDC1 has recently been implicated in tumorigenesis and tumor growth. In this study, we investigated its expression in cervical cancers and explored its function using gene knockdown approaches. We report here that NFBD1 expression is substantial increased in 24 of 39 cases (61.5%) of cervical cancer tissues at the mRNA level and in 35 of 60 cases (58.3%) at the protein level compared with the case matched normal tissues. Tumors with higher grade of malignancy tend to have higher levels of NFBD1 expression. By infecting cells with retroviruses expressing NFBD1 shRNA, we successfully knocked down NFBD1 expression in cervical cancer cell lines HeLa, SiHa, and CaSki. NFBD1 knockdown cells display significant growth inhibition, cell cycle arrest, higher apoptotic rate, and enhanced sensitivity to adriamycin. Furthermore, NFBD1 knockdown also inhibits the growth of HeLa cells in nude mice. Western blot analyses further revealed that NFBD1 knockdown induced Bax, Puma, and Noxa while down-regulating Bcl-2; it also up-regulated cytochrome C and activated caspases 3 and 9. Therefore, the function of NFBD1 may be involved in the CDC25C-CyclinB1/CDC2 pathway at the G2/M checkpoint, and the cytochrome C/caspase 3 apoptotic pathway. Since expression of NFBD1 seems to be related to the oncogenic potential of cervical cancer, and suppression of its expression can inhibit cancer cell growth both in vitro and in vivo, NFBD1 may be a potential therapeutic target in human cervical cancer.     

Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair

Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.     

The Viral Oncoprotein Tax Sequesters DNA Damage Response Factors by Tethering MDC1 to Chromatin

Infection with human T-cell leukemia virus induces cellular genomic instability mediated through the viral oncoprotein Tax. Here we present evidence that Tax undermines the cellular DNA damage response by sequestration of damage response factors. We show by confocal microscopy that Tax forms damage-independent nuclear foci that contain DNA-PK, BRCA1, and MDC1. Tax sequesters MDC1 to chromatin sites distinct from classic ionizing radiation-induced foci. The recruitment of MDC1 is competitive between the two foci. The N-terminal region of Tax is sufficient for foci localization, and the C-terminal half is critical for binding to MDC1 and recruitment of additional response factors. Tax expression and DNA damage response factor recruitment repressed the formation of ionizing radiation-induced Nbs1-containing foci. The Tax-induced “pseudo” DNA damage response results in phosphorylation and monoubiquitylation of H2AX, which is ablated by siRNA suppression of MDC1. These data support a model for virus-induced genomic instability in which viral oncogene-induced damage-independent foci compete with normal cellular DNA damage response.     

gammaH2AX and MDC1: anchoring the DNA-damage-response machinery to broken chromosomes

Higher-order chromatin structure presents a barrier to the recognition and repair of DNA lesions. Thus, cells must be equipped with mechanisms to surpass this natural obstacle. DNA damage induces histone H2AX phosphorylation by the phosphoinositide 3-kinase like kinases ATM, ATR and DNA-PKcs. H2AX phosphorylation contributes to DNA double-strand break repair but the mechanisms involved are not yet fully understood. In this review, we discuss recent advances in our understanding of how cells use the epigenetic mark of H2AX phosphorylation to dynamically link the DNA-damage-response machinery to broken chromosomes. In addition, we highlight potential regulatory mechanisms of H2AX phosphorylation and speculate about a central functional role of this post-translational histone modification at the interface of DNA repair, chromatin-structure modulation and cell-cycle checkpoint activation.     

Sumoylation of MDC1 is important for proper DNA damage response

In response to DNA damage, many DNA damage factors, such as MDC1 and 53BP1, redistribute to sites of DNA damage. The mechanism governing the turnover of these factors at DNA damage sites, however, remains enigmatic. Here, we show that MDC1 is sumoylated following DNA damage, and the sumoylation of MDC1 at Lys1840 is required for MDC1 degradation and removal of MDC1 and 53BP1 from sites of DNA damage. Sumoylated MDC1 is recognized and ubiquitinated by the SUMO-targeted E3 ubiquitin ligase RNF4. Mutation of the MDC1 Lys 1840 (K1840R) results in impaired CtIP, replication protein A, and Rad51 accumulation at sites of DNA damage and defective homologous recombination (HR). The HR defect caused by MDC1K1840R mutation could be rescued by 53BP1 downregulation. These results reveal the intricate dynamics governing the assembly and disassembly of DNA damage factors at sites of DNA damage for prompt response to DNA damage.     

Oligomerization of MDC1 protein is important for proper DNA damage response

Mediator of DNA damage checkpoint 1 (MDC1) plays an important role in the DNA damage response (DDR). MDC1 functions as a mediator protein and binds multiple proteins involved in different aspects of the DDR. However, little is know about the organization of MDC1 complexes. Here we show that ataxia telangiectasia, mutated (ATM) phosphorylates MDC1 at Thr-98 following DNA damage, which promotes its oligomerization. Oligomerization of MDC1 is important for the accumulation of MDC1 complex at the sites of DNA damage. Mutation of Thr-98 (T98A) would abolish its oligomerization and result in a defect in DNA damage checkpoint activation and increased sensitivity to irradiation. Taken together, these results suggest that the oligomerization of MDC1 plays an important role in DDR and help understand the formation of proteins complexes at the sites of DNA damage.     

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.