Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

ABSTRACT: Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA's 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA's kinetic cycle. Measurement of UvrA's steady-state ATPase activity shows it is stimulated upon binding DNA ( k_cat 0.71-1.07/s). Despite UvrA's ability to discriminate damage, we find UV-damaged DNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecul

SUBMITTER: Barnett JT 

PROVIDER: S-EPMC6355085 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature