title: Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease.
abstract: Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.
authors.affiliation: Environmental Toxicology Graduate Program, University of California, Riverside, California 92521-0403, USA.
authors.forename: Chunang
authors.initials: C
authors.lastname: Gu
authors.forename: Qibin
authors.initials: Q
authors.lastname: Zhang
authors.forename: Zhengguan
authors.initials: Z
authors.lastname: Yang
authors.forename: Yuesong
authors.initials: Y
authors.lastname: Wang
authors.forename: Yue
authors.initials: Y
authors.lastname: Zou
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D011740:Pyrimidine Dimers / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC