Abstract
The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide excision repair of a large variety of DNA damages. The damage recognition and incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent DNA helix-tracking activity associated with the UvrA2B1 complex. The latter activity leads to the generation of highly positively supercoiled DNA in the presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can contribute to the above reaction by abolishing the accumulation of highly positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision. Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to incise highly positively supercoiled uvDNA results from the failure of the formation of UvrAB-dependent obligatory intermediates associated with the DNA conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV endonuclease efficiently incises uvDNA regardless of its topological state. The in vitro topodynamic system proposed in this study may provide a simple model for studying a topological aspect of nucleotide excision repair and its interaction with other DNA topology-related processes in E. coli.
Highlights
The Uvr(A)BC endonuclease from Escherichia coli consists of an ensemble of the uvrA, uvrB, and uvrC gene products
It was found that highly positively supercoiled uvDNA generated by UvrA2B1 helix-tracking activity in the presence of only E. coli topo I is not incised by Uvr(A)BC, whereas it is efficiently incised by Micrococcus luteus UV endo
The results are discussed in terms of the influence of DNA topology on the process of Uvr(A)BC damage recognition and incision as well as in terms of the applicability of this in vitro topodynamic system for study of the topological aspect of NER and its interaction with other DNA topology-involved cellular processes
Summary
Proteins—The UvrA, UvrB, and UvrC proteins were purified according to previously published procedures [26, 27]. Preparation of Highly Positively Supercoiled DNA—The relaxed plasmid (100 –200 ng) pTZ18R (cold or 3H-labeled) was incubated with 400 ng each of UvrA and UvrB, and 400 ng of E. coli DNA topo I in the supercoiling buffer (see below) for 60 min at 37 °C. The relaxed (by calf thymus topo I) pTZ18R plasmid DNA (40 ng), containing approximately 24 cyclobutane pyrimidine dimers per circle, was incubated at 37 °C in supercoiling buffer (30 mM Tris-Cl, pH 7.5, approximately 100 nM KCl, 10 mM MgCl2, 1 mM DTT, and 4 mM ATP) with UvrA (200 ng) and UvrB (200 ng) in the presence of 80 ng of E. coli topo I. The data were quantified using a PhosphorImager (Fujix Bas 1000) and MacBas 2.0 software
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