Summary of complementation groups of UV-sensitive CHO cell mutants isolated by large-scale screening

Abstract

A summary is given for the lineage and complementation group assignments of 153 UV-sensitive mutants of the CHO AA8 cell line. The distribution of mutants among six complementation groups was highly non-random, with the great majority of the isolates belonging to groups 1 and 2. This asymmetry is consistent with the known hemizygosity of these two linked loci in CHO cells. The relative numbers of mutants induced in group 2 was found to depend greatly on the type of mutagen used. Mutagenesis with UV radiation, ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine and 7-bromomethylbenz[a]anthracene produced high frequencies of group 2 mutants. In contrast, ICR170 and ICR191, which are thought to produce mostly frameshift mutations, yielded very few mutants in group 2. These results are of particular importance in light of the recent finding that the human ERCC2 gene, which corrects group 2 mutants, has very strong homology with the yeast gene RAD3. RAD3 is an essential gene for viability in yeast, and the low recovery of group 2 mutants using the frameshift agents strongly suggests that frameshift mutations tend to be lethal in the hamster ERCC2 locus. Several mutagen-sensitive double mutants were isolated in two-step selections from EMS-, mitomycin C- or UV-sensitive parental cells, including the line UVU1, the first mammalian line with two mutations that affect UV sensitivity. The first mutation inactivated excision repair, and the second mutation appears to have affected some other recovery process. UVU1 should be useful for studying recovery processes that are separate from nucleotide excision repair.

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

“…XPG plasmid concentrations were determined by measurement of absorbance at 260 nm and verified by agarose gel electrophoresis and ethidium bromide staining. The XPG-deficient CHO cell line UV135 (16,23) was transfected with a mixture of 150 ng of luciferase plasmid, 150 ng of ␤-galactosidase plasmid (22), and 30 ng of wild-type or mutant XPG plasmid by calcium phosphate precipitation (16). For each XPG transfection set, three to six 60-mm dishes were used.…”

ERCC1/XPF limits L1 retrotransposition

“…This would imply that the "flap" [19]. ERCC1 and XPF were initially recognized as components of Nucleotide Excision Repair (NER) in both human and hamster cells with defects in repair after UV irradiation [20][21][22]. The specific biochemical role of the ERCC1/XPF heterodimer in NER is to nick the unwound DNA 5' of the lesion [23].…”

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

“…The UVsensitive cell line UV135 was derived from parental CHO AA8 cells as previously described (6). UV135 is deficient in the damage site incision step of ER (60).…”

“…Six proteins, including the uvrABC excinuclease complex, helicase II (uvrD), DNA polymerase I, and DNA ligase are the minimal necessary and sufficient components to excise the lesion containing oligonucleotide in vitro and to replace it by gap-filling DNA synthesis (22,55). In contrast, the genetic and biochemical evidence in eucaryotes now implicates more than 10 genes and proteins as crucial for ER (6,17,31). The increased complexity of eucaryotic ER is also evidenced by the general lack of resemblance of almost all known ER proteins of yeasts and mammals compared with those of E. coli.…”

“…Characterization of XP and CS genes was first made possible by human gene transfer into rodent CHO cell mutants (6). By using this method, human repair genes ERCC1, ERCC2 (XP-D), ERCC3 (XP-B), and ERCC6 (CS group B) have been sequenced and found to encode different deduced proteins (14,62,63,66,67).…”