Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.
UvrA, UvrB, and UvrC initiate nucleotide excision repair by incising a damaged DNA strand on each side of the damaged nucleotide. This incision reaction is substoichiometric with regard to UvrB and UvrC, suggesting that both proteins remain bound following incision and do not "turn over." The addition of only helicase II to such reaction mixtures turns over UvrC; UvrB turnover requires the addition of helicase II, DNA polymerase I, and deoxynucleoside triphosphates. Column chromatography and psoralen photocross-linking experiments show that following incision, the damaged oligomer remains associated with the undamaged strand, UvrB, and UvrC in a post-incision complex. Helicase II releases the damaged oligomer and UvrC from this complex, making repair synthesis possible; DNase I footprinting experiments show that UvrB remains bound to the resulting gapped DNA until displaced by DNA polymerase I. The specific binding of UvrB to a psoralen adduct in DNA inhibits psoralen-mediated DNA-DNA cross-linking, yet promotes the formation of UrvB-psoralen-DNA cross-links. The discovery of psoralen-UvrB photocross-linking offers the potential of active-site labeling.
Orren
D K
DK
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Selby
C P
CP
Hearst
J E
JE
Sancar
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004249:DNA Damage; D016172:DNA Fingerprinting; D004265:DNA Helicases; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005363:Ficusin / Q000494:pharmacology; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D005363:Ficusin