Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function.
The human ERCC3 gene, which corrects specifically the nucleotide excision repair defect in human xeroderma pigmentosum group B and cross-complements the repair deficiency in rodent UV-sensitive mutants of group 3, encodes a presumed DNA helicase that is identical to the p89 subunit of the general transcription factor TFIIH/BTF2. To examine the significance of the postulated functional domains in ERCC3, we have introduced mutations in the ERCC3 cDNA by means of site-specific mutagenesis and have determined the repair capacity of each mutant to complement the UV-sensitive phenotype of rodent group 3 cells. A conservative substitution of arginine for the invariant lysine residue in the ATPase motif (helicase domain I), six deletion mutations in the other helicase domains, and a deletion in the potential helix-turn-helix DNA-binding motif fail to complement the ERCC3 excision repair defect of rodent group 3 mutants, which implies that the helicase domains as well as the potential DNA-binding motif are required for the repair function of ERCC3. Analysis of carboxy-terminal deletions suggests that the carboxy-terminal exon may comprise a distinct determinant for the DNA repair function. In addition, we show that a functional epitope-tagged version of ERCC3 accumulates in the nucleus. Deletion of the putative nuclear location signal impairs neither the nuclear location nor the repair function, indicating that other sequences may (also) be involved in translocation of ERCC3 to the nucleus.
Ma
L
L
MGC-Laboratory for Molecular Carcinogenesis, Sylvius Laboratories, Leiden University, The Netherlands.
Westbroek
A
A
Jochemsen
A G
AG
Weeda
G
G
Bosch
A
A
Bootsma
D
D
Hoeijmakers
J H
JH
van der Eb
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002470:Cell Survival / Q000528:radiation effects; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D004307:Dose-Response Relationship, Radiation; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D015183:Restriction Mapping; D012377:Rodentia; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009838:Oligodeoxyribonucleotides; C078672:hay protein, Drosophila