Role of the two ATPase domains of Escherichia coli UvrA in binding non-bulky DNA lesions and interaction with UvrB.

The UvrA protein is the initial DNA damage-sensing protein in bacterial nucleotide excision repair and detects a wide variety of structurally unrelated lesions. After initial recognition of DNA damage, UvrA loads the UvrB protein onto the DNA. This protein then verifies the presence of a lesion, after which UvrA is released from the DNA. UvrA contains two ATPase domains, both belonging to the ABC ATPase superfamily. We have determined the activities of two mutants, in which a single domain was deactivated. Inactivation of either one ATPase domain in Escherichia coli UvrA results in a complete loss of ATPase activity, indicating that both domains function in a cooperative way. We could show that this ATPase activity is not required for the recognition of bulky lesions by UvrA, but it does promote the specific binding to the less distorting cyclobutane-pyrimidine dimer (CPD). The two ATPase mutants also show a difference in UvrB-loading, depending on the length of the DNA substrate. The ATPase domain I mutant was capable of loading UvrB on a lesion in a 50 bp fragment, but this loading was reduced on a longer substrate. For the ATPase domain II mutant the opposite was found: UvrB could not be loaded on a 50 bp substrate, but this loading was rescued when the length of the fragment was increased. This differential loading of UvrB by the two ATPase mutants could be related to different interactions between the UvrA and UvrB subunits.