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

Barnett, Jamie T., Kad, Neil M (2019) Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics. The FASEB Journal, 33 (1). ISSN 0892-6638. (doi:10.1096/fj.201800899R)

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 (kcat 0.71–1.07/s). Despite UvrA’s ability to discriminate damage,we find UV-damagedDNA 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 molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics.

Item Type: Article
DOI/Identification number: 10.1096/fj.201800899R
Uncontrolled keywords: cooperativity; fluorescence imaging; DNA repair; DNA tightropes
Subjects: Q Science
Q Science > QR Microbiology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: N. Kad
Date Deposited: 20 Sep 2018 15:32 UTC
Last Modified: 06 Jun 2019 13:28 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/69165 (The current URI for this page, for reference purposes)
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