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

Abstract

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.

ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability

“…After DNA damage, ATM is recruited to DSBs where it phosphorylates H2AX 1 , and then γ-H2AX serves as a docking platform for MDC1, which binds to γ-H2AX via its tBRCT domain 17 . Abrogation of the MDC1-γ-H2AX interaction disrupts IR-induced MDC1 foci formation and renders cells radiosensitive 45 . In this study, we observed the interaction between MDC1 and γ-H2AX was abolished by depletion of ID3.…”

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

“…This modification appears to promote the direct interaction between a minor portion of MDC1 molecules and RAP80, and the functional significance of this interaction is supported by a RAP80 delE81 point mutation, identified in familial breast cancer, that blocks the interaction [398]. This damage-independent interaction is required for the damage-dependent recruitment of RAP80 into nuclear foci [399] discussed in the next section. As a member of the MRN complex, NBS1 promotes both NHEJ [85] and HRR [400].…”

Reading, writing, and repair: the role of ubiquitin and the ubiquitin-like proteins in DNA damage signaling and repair

“…MDC1 also mediates recruitment of RAP80 to DNA damage foci. Although the E3 ligase remains unknown, ubiquitination of K1977 within the BRCT domain of MDC1 is required for recruitment of RAP80 to DNA DSBs (Strauss and Goldberg, 2011; Strauss et al, 2011). The K63-specific E2 Ubc13–Mms2 is required for RAP80 recruitment, implying that RAP80 is recruited to breaks through K63-linked ubiquitin chains (Strauss and Goldberg, 2011; Strauss et al, 2011).…”

“…Although the E3 ligase remains unknown, ubiquitination of K1977 within the BRCT domain of MDC1 is required for recruitment of RAP80 to DNA DSBs (Strauss and Goldberg, 2011; Strauss et al, 2011). The K63-specific E2 Ubc13–Mms2 is required for RAP80 recruitment, implying that RAP80 is recruited to breaks through K63-linked ubiquitin chains (Strauss and Goldberg, 2011; Strauss et al, 2011). Specifically, RAP80 is targeted to ubiquitin–SUMO hybrid chains through its SIM and two UIMs, and mutation of either the UIMs or the SIM in RAP80 decreases RAP80 recruitment to repair centers (Guzzo et al, 2012; Hu et al, 2012).…”