CONSTRUCTION OF A FUNCTIONAL CDNA CLONE OF THE HAMSTER ERCC2 DNA-REPAIR AND TRANSCRIPTION GENE

The complete hamster ERCC2 cDNA was constructed in a plasmid vector fr om clones of three overlapping reverse transcribed/polymerase chain re action amplified fragments using unique restriction enzyme recognition sites within the regions of overlap. This complete cDNA insert was th en closed into a mammalian expression vector, pcD2E, and tested for fu nction by the ability to confer UV resistance to the ERCC2 mutant CHO cell line UV5. Site-specific mutagenesis was used to introduce the G(3 47) --> A and G(1844) --> A changes resulting in the Cys116 --> Tyr an d Gly615 --> Glu mutations previously identified in UV5 and UVL-13 (al so an ERCC2 mutant CHO cell line), respectively. The 116(Tyr) and 615( Glu) plasmids each failed to confer UV resistance to UV5 or UVL-13 cel ls, respectively, demonstrating that the changes identified are indeed the causative mutations in UV5 and UVL-13.