Molecular cloning and biological characterization of the human excision repair gene ERCC-3.
In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).
Weeda
G
G
Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, Leiden, The Netherlands.
van Ham
R C
RC
Masurel
R
R
Westerveld
A
A
Odijk
H
H
de Wit
J
J
Bootsma
D
D
van der Eb
A J
AJ
Hoeijmakers
J H
JH
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects*; D005796:Genes; D015698:Genomic Library; D006801:Humans; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D014162:Transfection; D014466:Ultraviolet Rays
D000477:Alkylating Agents; D008937:Mitomycins; D016685:Mitomycin; D004247:DNA; D008741:Methyl Methanesulfonate