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.
Kadkhodayan
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S
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Salazar
E P
EP
Lamerdin
J E
JE
Weber
C A
CA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases; D004260:DNA Repair / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins; D005822:Genetic Vectors; D010957:Plasmids; D011506:Proteins / Q000235:genetics*; D014157:Transcription Factors; D014158:Transcription, Genetic; D051759:Xeroderma Pigmentosum Group D Protein
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein