Identification of E. coli uvrC protein.

Abstract

The excision of pyrimidine dimers from DNA is one of the major mechanisms of repair involving an initial endonucleolytic step followed by excision, resynthesis and ligation1. The enzymatic mechanism for endonucleolytic cleavage of UY light-damaged DNA by the Micrococcus luteus2 and Escherichia coli bacteriophage T4 (ref. 3) dimer-specific endonucleases involves two steps: cleavage of the N-glycosylic bond of the 5′-pyrimidine moiety of the pyrimidine dimer, followed by cleavage of the phosphodiester bond 3′ to the apyrimidinic site produced by the first step. Although E. coli requires functional uvrA, uvrB and uvrC genes to repair UV light-damaged DNA4, and enzymatic cleavage of damaged DNA requires the concerted action of at least three proteins5–8 the mechanism of catalysis by which the uvr-endonucleolytic system cleaves UV light-damaged DNA is unclear. We have constructed hybrid multicopy plasmids containing the uvr genes of E. coli9 to study the expression of amplified uvr genes. We have physically mapped uvrC on plasmid pGHY4211 (previously referred to as pGY4211 (ref. 9)) by interruption with the γδ sequence of F+ as described by Guyer10, and determined the minimum size for the uvrC gene to be 1.45 kilobase pairs. SDS-gel electrophoresis of radioiso-topically labelled plasmid proteins indicates that the uvrC+ gene product is a polypeptide of molecular weight (MW) 68,000. We estimate that wild-type E. coli may have as few as 10 copies of uvrC polypeptide per cell.