Ubiquitination of chromatin surrounding a double-stranded DNA break facilitates the recruitment of proteins that mediate the DNA damage response, adding another level of complexity to the regulation of DNA repair.
The DNA-damage response (DDR) signalling network mediates context-specific DNA repair, cell cycle checkpoint activation and/or apoptosis. DNA double-strand breaks (DSBs) cause DDR proteins to redistribute into dynamic, higher-order multiprotein assemblies on the surrounding chromatin. Adaptor proteins are crucial to build these assemblies in a spatial and temporal manner. This is achieved, in part, by hierarchical phosphorylations that are selectively recognized by downstream DDR proteins. Three separate papers, by Huen et al., Mailand et al. and Kolas et al., show that ubiquitylation of the chromatin that surrounds DSBs also contributes to orchestration of the DDR.
Once a DSB has been detected, the histone H2A variant H2AX surrounding the DSB is phosphorylated (termed H2AX). MDC1, an adaptor that binds directly to H2AX, is a crucial conductor of the DDR because it is required for the accumulation of other adaptor proteins, such as NBS1, 53BP1 and BRCA1. In all three studies, RNF8, a member of the RING-finger-containing nuclear factor family, was identified as a DDR protein that functions downstream of H2AX and MDC1 and interacts directly with MDC1 in response to DSBs. RNF8 contains an FHA domain, which binds phospho-Thr moieties, and a RING finger, which is common to E3 ubiquitin ligases. RNF8 binds to MDC1 through its FHA domain, and this interaction is dependent on MDC1 phosphorylation. Consistently, all three groups showed that MDC1 was phosphorylated in response to DSBs, and Kolas et al. showed that ATM (ataxia-telangiectasia mutated; a DDR kinase) was responsible.
So, what is the function of RNF8? Depletion of RNF8 from cells by small interfering RNA revealed that RNF8 was required for the accumulation of 53BP1 and BRCA1 and for the appearance of conjugated ubiquitin moieties surrounding DSBs. Mailand et al. demonstrated that adaptor assembly exhibited two distinct temporal stages whereby MDC1, RNF8 and NBS1 associate before 53BP1 and BRCA1. Mailand et al. also showed that ubiquitin was required for accumulation of the late adaptors, and all three groups showed that RNF8 (specifically the RING finger) was required for DSB-induced ubiquitylation, indicating that RNF8-dependent ubiquitylation is required for this temporal accumulation of late adaptor proteins. Kolas et al. and Huen et al. also showed that the E2 ubiquitylating enzyme UBC13 was equally required for accumulation of the late adaptors. Furthermore, Mailand et al. showed that RNF8 ubiquitylates the core histone H2A and, to a lesser extent, H2AX, and Huen et al. showed that RNF8–UBC13 ubiquitylates H2AX. Whether this ubiquitylation is necessary for the direct recruitment of DDR proteins or whether this serves to modify the chromatin microenvironment — thus indirectly leading to the association and retention of DDR proteins — remains unclear. However, all groups showed that RNF8-deficient cells were hypersensitive to ionizing radiation, and Huen et al. and Kolas et al. showed that RNF8 was required for DSB-induced G2–M checkpoint arrest (although Mailand et al. could not detect this).
Therefore, not only is RNF8 a bona fide new member of the DDR network, but further understanding of the hierarchical and temporal control of the early events of the DDR in response to DSBs has been achieved. Like phosphorylation, ubiquitylation might represent another level by which the DDR network can be intricately regulated, and this orchestration could be relevant to other types of DNA damage.
Gemma K. Alderton
References
Huen, M. S. Y. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell131, 901–914 (2007) | Article | PubMed |
Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell131, 887–900 (2007) | Article | PubMed |
Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science318, 1637–1640 (2007) | Article | PubMed |
H2AX). 