Ubiquitin-mediated recruitment of genome caretakers to DNA double-strand breaks

Introduction

A striking feature of the cellular response to DNA double strand breaks (DSBs) is the accumulation of proteins into large and microscopically discernible structures in the vicinity of the lesions – the so-called ionizing radiation induced foci (IRIF). This accumulation process is orchestrated by posttranslational histone modifications, which serve as affinity platforms for DNA damage response (DDR) proteins. The most proximal of these modifications is phosphorylation of the histone variant H2AX by the ATM kinase (γ-H2AX), which leads to the recruitment of MDC1 and its associated protein partners. One of these is the ubiquitin ligase RNF8, the activity of which initiates a complicated cascade of chromatin ubiquitylation events, which ultimately facilitate the recruitment of downstream DDR factors such as 53BP1, BRCA1 and RAD18.

DDR-associated chromatin ubiquitylation is an exquisitely complicated and highly regulated process that requires the sequential action of two ubiquitin ligases, RNF8 and RNF168. The latter targets histone H2A for ubiquitylation at K13 and K15, providing affinity sites for the downstream factors. While initially it was thought that RNF8 and RNF168 both targeted H2A-type core histones, recent advances have shown that RNF8 modifies unique substrates including linker histone H1 to provide a recruitment platform for RNF168 at sites of DNA damage.

Results and conclusions

In my talk, I will review the status of our ongoing efforts to understand the DDR-associated chromatin pathway. I will also report on our recent discovery of a new DDR factor, SCAI, with molecular links to both heterochromatin and NHEJ- and HR-associated components, and the potential to balance the activities of DSB repair pathways in the context of chromatin. SCAI is a novel HP1 interactor that accumulates at sites of DNA damage through two distinct binding mechanisms that simultaneously target the protein to both DNA-restricted HR repair foci and the surrounding chromatin compartment. These recruitment processes can be genetically uncoupled by depletion of either CtIP to prohibit DNA resection, or depletion of 53BP1, which interacts with and recruits SCAI to DSB-proximal chromatin regions. In a mouse model SCAI deficiency is associated with meiotic defects, and cells from these animals display delayed DSB repair kinetics in heterochromatic regions. Collectively our findings suggest that SCAI is a novel DDR factor that licenses effective DSB repair in heterochromatin.