Only a small proportion of the proteins that gather around a DNA double-strand break are actively involved in repairing the damage, Agarwal et al. suggest.
Double-strand breaks are repaired by homologous recombination, in which missing sections of DNA are replaced using sister chromatids as undamaged templates. Rad51 is the central catalyst of this process, working in combination with accessory factors like the DNA-dependent ATPase Rad54. Several functions have been proposed for Rad54, including modulating Rad51 binding on homologous DNA duplexes. But few studies have addressed Rad54's contribution to double-strand break repair in vivo.
Agarwal et al. generated cell lines expressing wild-type or ATPase-deficient Rad54 at endogenous levels. Cells that only expressed ATPase-dead Rad54 were as sensitive to DNA damage as cells that lacked Rad54 entirely, yet Rad51 accumulated rapidly at double-strand breaks as long as Rad54—even without its ATPase activity—was present. However, Rad54 remained at damage sites for longer if it lacked ATPase function, suggesting that ATP hydrolysis is required for the protein's dissociation from DNA.
The researchers estimated that between 100 and 600 Rad54 molecules normally accumulate at each spot of DNA damage. Only 10–60 of these molecules appear to be bound to the DNA, however, as photobleaching experiments revealed that mutations in Rad54's ATPase domain only immobilized 10% of the protein in each repair focus. Agarwal et al. don't yet know why so much extra Rad54 is recruited to double-strand breaks, but they plan to home in further on the molecules that actually perform the repair job by developing single-molecule imaging approaches.
