Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease

Yue Zou, Randall Walker, Heather Bassett, Nicholas E. Geacintov, Bennett Van Houten

Research output: Contribution to journalArticlepeer-review

Abstract

The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50- base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11- base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (K(d) ~-5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(β,γ- iminotriphosphate) or adenosine 5'-(β,γ-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.

Original languageEnglish (US)
Pages (from-to)4820-4827
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number8
DOIs
StatePublished - Feb 21 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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