The ATPase mechanism of UvrA(2) reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair

The UvrA(2) dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA(2) damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P(2ATP)-D(2ADP) species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA(2) to transition through a different species, P(2ADP)-D(2ADP). Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P(2ATP)-D(empty) and P(2ATP)-D(2ATP). Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA(2) forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA(2) utilizes ATP for lesion search, recognition and repair.