UV ENDONUCLEASE OF MICROCOCCUS-LUTEUS, A CYCLOBUTANE PYRIMIDINE DIMER-DNA GLYCOSYLASE ABASIC LYASE - CLONING AND CHARACTERIZATION OF THE GENE/

The gene of Micrococcus luteus UV endonuclease (cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase) was cloned and characterized. The cloned gene, whose product had a predicted molecular mass of 17,120 D a, was found to be capable of complementing the Escherichia coli uvrA6 mutation in vivo with respect to resistance to acetone-mediated molec ular photosensitization, a treatment producing exclusively cyclobutane pyrimidine dimers in DNA. It also generated a nicking activity specif ic for photosensitization-treated DNA by in vitro transcription/transl ation. When expressed in E. coil cells, the gene produced a protein st ructurally identical with UV endonuclease and possessing an activity c onsistent with cyclobutane pyrimidine dimer-DNA glycosylase/abasic lya se with respect to the effect of inhibitors and the site of the DNA ba ckbone scission. Furthermore, the UV endonuclease-deficient mutant DB7 was shown to regain the enzyme through transformation with the cloned gene. The deduced amino acid sequence of the gene product was at best 27% identical with that of endonuclease V of phage T4, an enzyme stri kingly similar to UV endonuclease in molecular and catalytic propertie s. Despite this marginal overall similarity in amino acid sequence, fo ur of the seven amino acid residues reported to be functionally import ant in the T4 enzyme were found to be conserved in the M. luteus enzym e. We propose that the gene be called uveA.