Repair Complex Assembly
The cascade of repair machinery assembly on a dsDNA break lesion occurs in a highly ordered manner. In mammalian cells, the primary damage is detected by by MRN (MRE11-RAD50-NBS1) complex. The MRN complex accumulates and activates the transducer kinase ATM which phosphorylates the histone variant H2AX. The mediator protein MDC1 then binds ɣ-H2AX and recruits additional copies of MRN and ATM, leading to spread of the repair machinery along the chromosome. MDC1 also recruits ubiquitin ligase activities (ex. RNF8) and other chromatin modification and remodelling complexes which subsequently recruit downstream factors such as 53BP1 and BRAC1. DNA is then resected to ssDNA which is recognized and bound by replication protein A (RPA). Bound RPA recruits ATRIP, followed by ATR which interacts with both of the aformentioned proteins. Both the ATM- and ATR- dependent branches of the pathway independently or in concert activate the checkpoint kinases CHK1 and CHK2.
DNA Damage Foci
Recruitment and accumulation of the DNA-repair factors at a site of DNA damage creates a macroscopically discernable focus which spreads up to one megabase from the break. DNA damage foci reveal that different repair factors exhibit distinct patterns of association with the DNA lesion. The recombination factors RAD51 and RAD52, the ATR kinase and its interacting partners ATRIP and RPA, and the DNA clamp proteins RAD17 and RAD9 are restricted to a small area around the DSB which corresponds mainly to stretches of ssDNA to which these proteins bind. Other factors, such as the MRN complex, MDC1, ATM, 53BP1 and BRCA1 spread up to one megabase away. Damage foci are thought to serve the purpose of DNA-damage signal amplification via recruitment of multiple signalling kinases to sites of damage. Downstream effectors of the DNA damage response (DDR), such as CHK1, CHK2, p53, and CDC25, do not visibly accumulate at damage foci, suggesting these factors interact transiently with sites of damage, whereupon they are activated before spreading to the entire nucleus.
(c): Microstructure of DNA damage repair foci.
(d): The center of the focus contains resected ssDNA which is bound by a specific set of factors that restore normal chromatin structure
(e): Regions that flank the DSB are occupied by a distinct set of proteins which spread up to one megabase away from the break. These factor are involved in spreading and amplifying the DDR signal.