84 research outputs found

    MDC1: The art of keeping things in focus

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    The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures—usually termed DNA damage foci—is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structure

    Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells

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    Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells

    Spectrum of topics for world congresses and other activities of the International Society for Physical and Rehabilitation Medicine (ISPRM) : a first proposal

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    Background: One of the objectives of the International Society for Physical and Rehabilitation Medicine is to improve the continuity of World Congresses. This requires the development of an abstract topic list for use in congress announcements and abstract submissions. Methods: An abstract topic list was developed on the basis of the definitions of human functioning and rehabilitation research, which define 5 main areas of research (biosciences in rehabilitation, biomedical rehabilitation sciences and engineering, clinical Physical and Rehabilitation Medicine (PRM) sciences, integrative rehabilitation sciences, and human functioning sciences). For the abstract topic list, these research areas were grouped according to the proposals of congress streams. In a second step, the first version of the list was systematically compared with the topics of the 2003 ISPRM World Congress. Results: The resulting comprehensive abstract topic list contains 5 chapters according to the definition of human functioning and rehabilitation research. Due to the high significance of clinical research, clinical PRM sciences were placed at the top of the list, comprising all relevant health conditions treated in PRM services. For congress announcements a short topic list was derived. Discussion: The ISPRM topic list is sustainable and covers a full range of topics. It may be useful for congresses and elsewhere in structuring research in PRM

    4′-Acylated thymidine 5′-triphosphates: a tool to increase selectivity towards HIV-1 reverse transcriptase

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    4′-Acylated thymidines represent a new class of DNA chain terminators, since they have been shown to act as post-incorporation chain-terminating nucleotides despite the presence of a free 3′-hydroxyl group. Here, we describe the action of the 4′-acetyl- (MeTTP) and 4′-propanoylthymidine 5′-triphosphate (EtTTP) on HIV-1 reverse transcriptase in RNA- and DNA-dependent DNA synthesis and on DNA synthesis catalyzed by the cellular DNA polymerases α, β, δ and ε. MeTTP exhibits a high selectivity towards HIV-1 reverse transcriptase. By the use of the bulkier propanoyl group as the 4′-substituent of the nucleoside 5′-triphosphate, selectivity towards HIV-1 reverse transcriptase could be increased without affecting substrate efficiency. Thus, 4′-modifications may serve as a tool to increase selectivity towards HIV-1 reverse transcriptas

    Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

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    The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin

    NBS1 promotes the endonuclease of the MRE11-RAD50 complex by sensing CtIP phosphorylation

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    DNA end resection initiates DNA break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step by MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its Xrs2 homologue from Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in high eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP promotes MRE11: a phosphorylation-dependent mode through NBS1, and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection in absence of the FHA and BRCT domains of NBS1 occurs in vivo. Collectively, our data suggest that NBS1 restricts the MRE11- RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism

    The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

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    The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased radio-sensitivity, micronuclei formation and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex

    The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

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    Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damag
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