The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light - PubMed (original) (raw)

The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light

P E Gibbs et al. Proc Natl Acad Sci U S A. 2000.

Abstract

In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase zeta. As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol zeta, and here report the identification and sequence of a human homolog of yeast REV1. This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5' rapid amplification of cDNA ends. The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein. The proteins share two amino-terminal regions of approximately 100 residues with 41% and 20% identity, a region of approximately 320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical. Human cells expressing high levels of an hREV1 antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA. However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (P = 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.

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Figures

Figure 1

Figure 1

(A) Sequence of the translation product of hREV1 mRNA. The underlined sequences numbered I–IX are possible sequence motifs, and the highlighted residues in motifs I, III, and VI are known to be important for function. (B) Depiction of regions of similarity to C. elegans and D. melanogaster sequences (open boxes) together with the location of the predicted motifs. The darker filled boxes indicate the location of motifs III and VI.

Figure 2

Figure 2

Schematic representation of the alignment of hREV1 and yeast REV1 protein sequences. Regions with significant identity are shaded, and the percent identity is indicated.

Figure 3

Figure 3

Northern blot analysis of the level of expression of hREV1 antisense RNA in the cell strains. RNA extracted from the two derivative cell strains, 7AGM-17C and 7AGM-12B, which had been transfected with plasmid pR1P27-AS containing the 4,117-bp sequence of the hREV1 RNA in an antisense orientation, and from their nontransfected parental cell strain, MSU-1.2-7AGM, and analyzed for expression of hREV1 antisense RNA (hREV1 AS RNA) and/or the endogenous hREV1 sense RNA. The latter mRNA, which is ≈4.4 kbp in length, can be seen as a faint band just above the antisense band in the first two lanes, and in the third lane. The lower band, approximately 1.4 kbp in length, is the endogenous HPRT mRNA, which was used to normalize the amount of RNA loaded per lane. There was no significant difference between the two derivative cell strains in the level of expression of the antisense RNA.

Figure 4

Figure 4

Percent survival (A) and frequencies of UV-induced 6-thioguanine resistant mutants (B) in the parent cell strain 7AGM and the hREV1 antisense RNA-expressing cell strains 7AGM-17C-R1 and 7AGM-12B-R1, plotted against fluence of 254 nm UV. The data are the average of results from four experiments with the parent strain, 7AGM; three experiments with 7AGM-17C-R1 cells; and two experiments with 7AGM-12B-R1cells. The background frequencies of mutants, which have been subtracted to obtain the frequencies induced above the background frequency by UV (23), are cited in the text. The mutant frequencies observed for untreated control and each UV fluence were corrected for the cloning efficiency of the cells determined after an 8-day expression period, i.e., at the time they were plated into selection medium containing 6-thioguanine. The average cloning efficiencies for control and UV-irradiated cells were: 7AGM cells, 33.3% ± 1.4% (SEM for four independent experiments); 7AGM-17C-R1 cells, 26.7 ± 1.3% (SEM for three independent experiments); and 7AGM-12B-R1 cells, 31.8% ± 0.9% (SEM for two independent experiments).

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