The expression of the Escherichia coli uvrA gene in human cells

Abstract

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously.

The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt⁺ colonies were selected, and cell lines esttablished. Several times were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95 000-dalton UvrA-related protein. The expression of uvrA was found to be 50–100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the “mammalian” UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA⁺ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA⁺ cell lines were examined for their UV_resistant phenotye and not unexpectedly were found not to be reverted to a state of repair proficiency.