Cloning and characterization of the human DNA-excision repair gene ERCC-1

It is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship between DNA damage and carcinogenesis. Many attempts to complement the genetic defect in XP by genomic DNA transfer have failed which is most likely due to the poor transfectability of human cells. In contrast to XP cells, UV- sensitive Chinese hamster ovary (CHO) mutants are more suitable for genomic transfection which renders it possible to isolate the human excision repair cross complementing (ERCC) genes that correct the CHO mutations. The isolation of these genes opens the possibility to unravel the complex mechanism of DNA excision repair. In addition, it is still hoped that part of the spectra of rodent and human UV- sensitive cells overlap. In that case the cloning of ERCC-genes by genomic transfections with CHO mutants might at the same time aid the elucidation of the genetic defects in XP. The experimental work described in this dissertation concerns the cloning and chara.cterization of the first human DNA repair gene, designated ERCC-1, which complements the UV and mitomycin-C sensitivity of CHO mutants of complementation group 1. The ERCC-1 gene was cloned after cotransfection of high molecular weight human DNA with a dominant marker Ecogpt to CHO mutant 43-3B. The linked transfer of the ERCC-1 and the Ecogpt gene in the second round of transfection a.llowed the isolation of the ERCC-1 gene from a genomic cosmid library using a probe from the dominant marker. Correction of the repair deficient phenotype of 43-3B cells by transfection of cosmid 43-34 confirmed the cloning of ERCC-1 (Appendix paper 1). Using single copy probes from cosmid 43-34 the human ERCC-1 eDNA was isolated and in subsequent experiments human ERCC-1 eDNA probes allowed the cloning of the mouse ERCC-1 gene and eDNA. The results on the characterization of the human and murine ERCC-1 genes are described in Appendix papers II, III and IV.