A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

Abstract

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...

Functional Analysis of p53 Binding under Differential Stresses

“…RNA was harvested from p53 ϩ/ϩ and p53 Ϫ/Ϫ HCT116 cells after exposure to 0.02% O 2 or 0.3 g/ml adriamycin and assayed by qRT-PCR. We found that REV3L, the human homolog of the catalytic subunit of DNA polymerase (18), is induced by DNA damage (Fig. 4 and 5B).…”

“…8). An induced gene of particular interest was REV3L, the catalytic subunit of DNA polymerase (18). Although p53-dependent induction is not as high as it is for genes like p21, a two-to threefold increase of a key DNA repair enzyme may have profound consequences.…”

UV-B Radiation Induces Epithelial Tumors in Mice Lacking DNA Polymerase η and Mesenchymal Tumors in Mice Deficient for DNA Polymerase ι

“…Several studies using small interfering RNA on mammalian TLS polymerases have shown that Rev1 and Rev3, but not Pol , are clearly required in UV-induced mutagenesis (9,19,20,26,35). However, it is currently unknown what kind of mechanism is involved in the observed hypermutability of XP-V cells.…”

Translesion synthesis in mammalian cells

“…Rev1, Rev3 and Rev7 were originally identified in S. cerevisae as being required for mutations induced by most DNA damaging agents [15]. A similar requirement has also been found in human cells [16,17]. This implies that they are involved in TLS, often inserting the "wrong" bases.…”