UvrAB activity at a damaged DNA site: is unpaired DNA present?
To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp. Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct. Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites. We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs.
Gordienko
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Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520-8040, USA.
Rupp
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D015073:2-Acetylaminofluorene; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides
D001426:Bacterial Proteins; D018736:DNA Adducts; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C058093:UvrB protein, E coli; D015073:2-Acetylaminofluorene; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases