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 from clones of three overlapping reverse transcribed/polymerase chain reaction amplified fragments using unique restriction enzyme recognition sites within the regions of overlap. This complete cDNA insert was then cloned into a mammalian expression vector, pcD2E, and tested for function by the ability to confer UV resistance to the ERCC2 mutant CHO cell line UV5. Site-specific mutagenesis was used to introduce the G347-->A and G1844-->A changes resulting in the Cys116-->Tyr and Gly615-->Glu mutations previously identified in UV5 and UVL-13 (also an ERCC2 mutant CHO cell line), respectively. The 116Tyr and 615Glu plasmids each failed to confer UV resistance to UV5 or UVL-13 cells, respectively, demonstrating that the changes identified are indeed the causative mutations in UV5 and UVL-13.