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

Melander F, Bekker-Jensen S, Falck J, Bartek J, Mailand N, Lukas J - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Biology and 2Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.

ABSTRACT
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.

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A hypothetical model of the MDC1–MRN interaction before and after DNA damage. In undamaged cells, CK2 targets the SDT-rich N terminus of MDC1. Phosphorylated SDTs are recognized by the FHA domain of NBS1 and allow dynamic interaction between MDC1 and the MRN complex (top). DSBs trigger the phosphorylation of H2AX (middle) followed by its recognition by the MDC1-associated BRCT domains (bottom). The resulting concentration of phosphorylated SDT repeated in the DSB-flanking chromatin allows immediate recruitment of the MRN complex to this compartment. CK2 (potentially together with another acidophilic kinase; indicated by an asterisk) may then stimulate MRN retention at the DSB sites by phosphorylating additional SDT repeats. Alternatively, the SDT-associated phosphates might be stabilized by inhibition of a DSB-associated protein phosphatase (PPase). Both mechanisms are compatible with a dynamic MRN exchange between distinct DSB-generated subcompartments but prevent its dilution in the undamaged nucleoplasm. See Discussion for an additional explanation.
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fig8: A hypothetical model of the MDC1–MRN interaction before and after DNA damage. In undamaged cells, CK2 targets the SDT-rich N terminus of MDC1. Phosphorylated SDTs are recognized by the FHA domain of NBS1 and allow dynamic interaction between MDC1 and the MRN complex (top). DSBs trigger the phosphorylation of H2AX (middle) followed by its recognition by the MDC1-associated BRCT domains (bottom). The resulting concentration of phosphorylated SDT repeated in the DSB-flanking chromatin allows immediate recruitment of the MRN complex to this compartment. CK2 (potentially together with another acidophilic kinase; indicated by an asterisk) may then stimulate MRN retention at the DSB sites by phosphorylating additional SDT repeats. Alternatively, the SDT-associated phosphates might be stabilized by inhibition of a DSB-associated protein phosphatase (PPase). Both mechanisms are compatible with a dynamic MRN exchange between distinct DSB-generated subcompartments but prevent its dilution in the undamaged nucleoplasm. See Discussion for an additional explanation.

Mentions: Given these findings, the new data presented in this study, and similar results reached by Spycher et al. (see p. 227 of this issue), our current model describing the mechanism of MRN chromatin retention is as follows. In undamaged nuclei (Fig. 8, top), CK2 phosphorylates the SDT repeats of MDC1. Combined with the ability of the NBS1-FHA domain to recognize phosphorylated SDTs, this allows constitutive yet highly dynamic MDC1–MRN interaction, which remains amenable to further regulation and prevents sequestration of any of these factors in rigid aggregates. The latter feature could be crucial to allow the rapid recognition of DNA breaks by the MRN complex, a process that is independent of MDC1 (see Introduction). After DSB generation (Fig. 8, bottom), the most proximal chromatin modification includes phosphorylation of the H2AX C terminus (γ-H2AX). Although several DSB regulators contain phosphate interaction motifs and domains, the MDC1-BRCT domain appears to interact with γ-H2AX with the highest affinity (Stucki et al., 2005), resulting in a rapid coating of the γ-H2AX–primed chromatin by MDC1. At this point, the MDC1 N terminus becomes important for the ensuing events on the DSB-flanking chromatin (Fig. 8, bottom). We propose that constitutive phosphorylation of the SDT repeats (translated to a continuous generation of binding sites for the NBS1-FHA domain) is likely the most efficient means to avoid any delay in recruiting MRN to the modified chromatin rapidly evolving around the incipient DBS lesions. Indeed, integration of a constitutive signal is consistent with the simultaneous recruitment of MDC1 and NBS1 to DSBs (Lukas et al., 2004a; Mailand et al., 2007) and suggests that in vertebrates, the SDT repeats of MDC1 coevolved with MRN to prime the latter for the fastest possible arrival at the sites of harmful chromosomal lesions.


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

Melander F, Bekker-Jensen S, Falck J, Bartek J, Mailand N, Lukas J - J. Cell Biol. (2008)

A hypothetical model of the MDC1–MRN interaction before and after DNA damage. In undamaged cells, CK2 targets the SDT-rich N terminus of MDC1. Phosphorylated SDTs are recognized by the FHA domain of NBS1 and allow dynamic interaction between MDC1 and the MRN complex (top). DSBs trigger the phosphorylation of H2AX (middle) followed by its recognition by the MDC1-associated BRCT domains (bottom). The resulting concentration of phosphorylated SDT repeated in the DSB-flanking chromatin allows immediate recruitment of the MRN complex to this compartment. CK2 (potentially together with another acidophilic kinase; indicated by an asterisk) may then stimulate MRN retention at the DSB sites by phosphorylating additional SDT repeats. Alternatively, the SDT-associated phosphates might be stabilized by inhibition of a DSB-associated protein phosphatase (PPase). Both mechanisms are compatible with a dynamic MRN exchange between distinct DSB-generated subcompartments but prevent its dilution in the undamaged nucleoplasm. See Discussion for an additional explanation.
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Related In: Results  -  Collection

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fig8: A hypothetical model of the MDC1–MRN interaction before and after DNA damage. In undamaged cells, CK2 targets the SDT-rich N terminus of MDC1. Phosphorylated SDTs are recognized by the FHA domain of NBS1 and allow dynamic interaction between MDC1 and the MRN complex (top). DSBs trigger the phosphorylation of H2AX (middle) followed by its recognition by the MDC1-associated BRCT domains (bottom). The resulting concentration of phosphorylated SDT repeated in the DSB-flanking chromatin allows immediate recruitment of the MRN complex to this compartment. CK2 (potentially together with another acidophilic kinase; indicated by an asterisk) may then stimulate MRN retention at the DSB sites by phosphorylating additional SDT repeats. Alternatively, the SDT-associated phosphates might be stabilized by inhibition of a DSB-associated protein phosphatase (PPase). Both mechanisms are compatible with a dynamic MRN exchange between distinct DSB-generated subcompartments but prevent its dilution in the undamaged nucleoplasm. See Discussion for an additional explanation.
Mentions: Given these findings, the new data presented in this study, and similar results reached by Spycher et al. (see p. 227 of this issue), our current model describing the mechanism of MRN chromatin retention is as follows. In undamaged nuclei (Fig. 8, top), CK2 phosphorylates the SDT repeats of MDC1. Combined with the ability of the NBS1-FHA domain to recognize phosphorylated SDTs, this allows constitutive yet highly dynamic MDC1–MRN interaction, which remains amenable to further regulation and prevents sequestration of any of these factors in rigid aggregates. The latter feature could be crucial to allow the rapid recognition of DNA breaks by the MRN complex, a process that is independent of MDC1 (see Introduction). After DSB generation (Fig. 8, bottom), the most proximal chromatin modification includes phosphorylation of the H2AX C terminus (γ-H2AX). Although several DSB regulators contain phosphate interaction motifs and domains, the MDC1-BRCT domain appears to interact with γ-H2AX with the highest affinity (Stucki et al., 2005), resulting in a rapid coating of the γ-H2AX–primed chromatin by MDC1. At this point, the MDC1 N terminus becomes important for the ensuing events on the DSB-flanking chromatin (Fig. 8, bottom). We propose that constitutive phosphorylation of the SDT repeats (translated to a continuous generation of binding sites for the NBS1-FHA domain) is likely the most efficient means to avoid any delay in recruiting MRN to the modified chromatin rapidly evolving around the incipient DBS lesions. Indeed, integration of a constitutive signal is consistent with the simultaneous recruitment of MDC1 and NBS1 to DSBs (Lukas et al., 2004a; Mailand et al., 2007) and suggests that in vertebrates, the SDT repeats of MDC1 coevolved with MRN to prime the latter for the fastest possible arrival at the sites of harmful chromosomal lesions.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Biology and 2Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.

ABSTRACT
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.

Show MeSH