Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II.
Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.
Tomer
G
G
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Cohen-Fix
O
O
O'Donnell
M
M
Goodman
M
M
Livneh
Z
Z
D000251:Adenosine Triphosphatases / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology*; D002474:Cell-Free System; D004249:DNA Damage; D004265:DNA Helicases; D011088:DNA Ligases / Q000502:physiology; D004256:DNA Polymerase I / Q000502:physiology; D004257:DNA Polymerase II / Q000502:physiology*; D004258:DNA Polymerase III / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000502:physiology; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D016296:Mutagenesis / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000528:radiation effects; D013014:SOS Response, Genetics / Q000502:physiology*; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D011088:DNA Ligases