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 laggin g strand during DNA replication. Escherichia coli cells completely lacking the poll enzyme are viable as long as they are grown on minimal medium. Her e we show that viability is fully dependent on the presence of functional U vrA, UvrB, and UvrD (helicase II) proteins but does mt require UvrC. In con trast, Delta polA tells grow even better when the uvrC gene has been delete d. Apparently UvrA, UvrB, and UvrD are needed in a replication backup syste m that replaces the PolI function, and UvrC interferes with this alternativ e replication pathway, With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity tha t on damaged DNA is responsible for the 3' incision reaction. Specific muta nts of UvrA and UvrB were also studied for their capacity to support the po ll-independent replication. Deletion of the UvrC-binding domain of UvrB res ulted in a phenotype similar to that caused by deletion of the uvrC gene, s howing that the inhibitory incision activity of UvrC is mediated via bindin g 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 invi ability in combination with the Delta polA mutation. Apparently the N-termi nal zinc-binding domain of UvrA has specifically evolved for a function out side DNA repair. A model for the function of the UvrA, UvrB, and UvrD prote ins in the alternative replication pathway is discussed.