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

2008 ◽  
Vol 181 (2) ◽  
pp. 227-240 ◽  
Author(s):  
Christoph Spycher ◽  
Edward S. Miller ◽  
Kelly Townsend ◽  
Lucijana Pavic ◽  
Nicholas A. Morrice ◽  
...  
Keyword(s):  
Nijmegen Breakage Syndrome ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Interfering Rna

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.

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

2008 ◽  
Vol 181 (2) ◽  
pp. 213-226 ◽  
Author(s):  
Fredrik Melander ◽  
Simon Bekker-Jensen ◽  
Jacob Falck ◽  
Jiri Bartek ◽  
Niels Mailand ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Nijmegen Breakage Syndrome ◽  
Local Concentration ◽  
Dna Double Strand Breaks ◽  
Phosphate Binding ◽  
Chromosomal Breakage ◽  
Mrn Complex ◽  
N Terminus

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.

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals The internal region of CtIP negatively regulates DNA end resection

2020 ◽  
Vol 48 (10) ◽  
pp. 5485-5498 ◽  
Author(s):  
Sean Michael Howard ◽  
Ilaria Ceppi ◽  
Roopesh Anand ◽  
Roger Geiger ◽  
Petr Cejka
Keyword(s):  
Homologous Recombination ◽  
Regulatory Function ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Internal Region ◽  
Dna End Resection ◽  
End Resection

Abstract DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11–RAD50–NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM–DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350–600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN–CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550–600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

scholarly journals A Survey of Reported Disease-Related Mutations in the MRE11-RAD50-NBS1 Complex

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1678 ◽  
Author(s):  
Samiur Rahman ◽  
Marella D. Canny ◽  
Tanner A. Buschmann ◽  
Michael P. Latham
Keyword(s):  
Genomic Stability ◽  
Nijmegen Breakage Syndrome ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Response Factors ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Damage Response ◽  
Biochemical Level

The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease. Here, we discuss, in a structural and biochemical context, mutations in each of the three MRN components that have been associated with diseases such as ataxia telangiectasia-like disorder (ATLD), Nijmegen breakage syndrome (NBS), NBS-like disorder (NBSLD) and certain types of cancers. Overall, deepening our understanding of disease-causing mutations of the MRN complex at the structural and biochemical level is foundational to the future aim of treating diseases associated with these aberrations.

scholarly journals Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation

2008 ◽  
Vol 29 (4) ◽  
pp. 1000-1006 ◽  
Author(s):  
Christine S. Vissinga ◽  
Tiong C. Yeo ◽  
Sarah Warren ◽  
James V. Brawley ◽  
Jennifer Phillips ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Nuclear Export Signal ◽  
Nijmegen Breakage Syndrome ◽  
Dna Double Strand Breaks ◽  
Gene Encoding ◽  
Mrn Complex ◽  
Nbn Gene

ABSTRACT Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

scholarly journals POGZ modulates the DNA damage response in a HP1-dependent manner

2021 ◽  
Author(s):  
John Heath ◽  
Estelle Simo Cheyou ◽  
Steven Findlay ◽  
Vincent Luo ◽  
Edgar Pinedo Carpio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Genome Stability ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Humoral Immune ◽  
Damage Response

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonic lethal and Pogz haplo-insufficiency (Pogz+/Δ) results in a developmental delay, a deficit in intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/Δ mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

scholarly journals HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

2001 ◽  
Vol 21 (5) ◽  
pp. 1854-1865 ◽  
Author(s):  
Caitlin Hall ◽  
David M. Nelson ◽  
Xiaofen Ye ◽  
Kayla Baker ◽  
James A. DeCaprio ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ectopic Expression ◽  
Cyclin A ◽  
S Phase ◽  
Dependent Manner ◽  
Sequence Motifs ◽  
Histone Gene Expression ◽  
Phase Progression

ABSTRACT Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.

scholarly journals Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

2010 ◽  
Vol 207 (5) ◽  
pp. 983-997 ◽  
Author(s):  
Li Li ◽  
Marie-Jo Halaby ◽  
Anne Hakem ◽  
Renato Cardoso ◽  
Samah El Ghamrasni ◽  
...  
Keyword(s):  
Dna Damage ◽  
Class Switch Recombination ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Class Switch ◽  
Physiological Processes ◽  
Increased Sensitivity

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8.

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