To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase , a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an aminoterminal region of Ϸ340 residues, 39% identical in a carboxylterminal region of Ϸ850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol type; the carboxylterminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol among all polymerases in the GenBank database, and is different from the human ␣, ␦, and enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.In budding yeast, Saccharomyces cerevisiae, virtually all DNA damage-induced mutagenesis and a major fraction of spontaneous mutagenesis depends on the activity of the REV3 (scREV3) gene (1-4). The scREV3 locus encodes the catalytic subunit of DNA polymerase , which contains at least one other subunit, the product of the REV7 gene (5). Pol , together with the REV1 gene product and possibly other proteins, is thought to carry out translesion replication; that is, replication past DNA lesions that block chain elongation by the normal replicases (2, 5). Translesion replication is regulated, in a way that is still poorly understood, by the RAD6 and RAD18 genes, which encode an E2 ubiquitin conjugase and a DNA binding protein with which the conjugase associates, respectively (6).Although budding yeast is often a good model for other eukaryotes, it is not yet known whether similar proteins and mechanisms exist in other members of this group. Information of this kind from humans might be particularly useful, in view of its possible relevance to understanding genotoxic hazards and cancer. We have therefore conducted a search for human homologs of the scREV genes, as a preliminary step to investigating the enzymological basis of translesion replication in Homo sapiens. In this paper we report the isolation and sequence of a human homolog of scREV3 and evidence from human cells expressing an hsREV3 antisense mRNA fragment that this gene performs a function similar to that of its yeast counterpart. MATERIALS AND METHODScDNA Synthesis, 5 Rapid Amplification of cDNA Ends (RACE), and Cloning. Unless otherwise noted, the materials used were obtained fro...

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369 -26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.Oligonucleotides can recognize both nucleic acids and proteins with a high degree of specificity. This is a major reason why they have been widely investigated as potential therapeutic agents for cancer, viral infections, and inflammatory diseases. Oligonucleotides can achieve target recognition by sequence-specific interactions with nucleic acids or proteins such as in the antisense, antigene, or decoy approaches (1-4). Alternatively, target recognition can be due to the specific threedimensional structure of an oligonucleotide, as in the aptamer approach (5, 6). These aptameric oligonucleotides often contain secondary structure elements such as hairpins or G-quartets. The formation of G-quartet structures is also thought to contribute to non-antisense growth inhibitory effects of G-rich phosphodiester and phosphorothioate oligonucleotides (7-9).Recently, we reported (9) on a novel class of phosphodiester G-rich oligonucleotides (GROs) 1 that could strongly inhibit the in vitro proliferation of tumor cells derived from prostate, breast, and cervical carcinomas. The antiproliferative GROs were able to form stable secondary structures consistent with G-quartet format...

In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase . 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 , 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 Ϸ100 residues with 41% and 20% identity, a region of Ϸ320 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.