In vitro UV mutagenesis associated with nucleotide excision-repair gaps in Escherichia coli.
Using a cell-free system for UV mutagenesis we have recently shown that extracts prepared from Escherichia coli cells promote a UV mutagenesis pathway that depends on the uvrABC repair genes independent of DNA replication (type II UV mutagenesis; Cohen-Fix, O., and Livneh, Z. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3300-3304). Type II UV mutagenesis was defective also in extracts prepared from a uvrD strain. These deficiencies were complemented by adding purified UvrA, UvrB, UvrC, or UvrD proteins to the respective cell extracts. The Uvr proteins act at an early stage in the process, probably preparing a premutagenic single-stranded DNA gap, which subsequently serves as a substrate for the mutagenic reaction. Type II UV mutagenesis was not dependent on DNA polymerases I or on DNA polymerase II, but it was dependent on DNA polymerase III. Thus, similar to the in vivo situation, only DNA polymerase III is essential for UV mutagenesis. Antibodies against the beta subunit of DNA polymerase III holoenzyme inhibited DNA replication but not UV mutagenesis. Thus, the processivity subunit of the holoenzyme is not required for type II UV mutagenesis, in agreement with a mechanism involving filling-in of short single-stranded DNA gaps.
Cohen-Fix
O
O
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Livneh
Z
Z
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D000906:Antibodies / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004249:DNA Damage; D004265:DNA Helicases; D004256:DNA Polymerase I / Q000378:metabolism / Q000528:radiation effects; D004257:DNA Polymerase II / Q000378:metabolism / Q000528:radiation effects; D004258:DNA Polymerase III / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004261:DNA Replication / Q000528:radiation effects; D004269:DNA, Bacterial / Q000235:genetics / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000528:radiation effects*; D005838:Genotype; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014466:Ultraviolet Rays
D000906:Antibodies; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; 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; C506746:UvrD protein, E coli; D004265:DNA Helicases