Role of the Escherichia coli nucleotide excision repair proteins in DNA replication.
DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, delta polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the delta polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.
Moolenaar
G F
GF
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
Moorman
C
C
Goosen
N
N
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D008957:Models, Genetic; D016296:Mutagenesis; D010957:Plasmids; D014161:Transduction, Genetic
D001426:Bacterial Proteins; 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; C506746:UvrD protein, E coli; D004265:DNA Helicases