Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed inEscherichia coli

Abstract

Nucleotide excision repair in humans involves the coordinated actions of 8 - 1 0 proteins. To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins. Towards this goal we have expressed in E.coli two of the 8 genes known to be essential for the excision reaction. XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography. The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containingsubstrate.