Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene.
In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.
Clark
Denise R
DR
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA.
Zacharias
Wolfgang
W
Panaitescu
Luminita
L
McGregor
W Glenn
WG
D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005787:Gene Frequency; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D016296:Mutagenesis / Q000528:radiation effects*; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D007041:Hypoxanthine Phosphoribosyltransferase; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D016337:RNA, Catalytic; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases; C429004:REV1 protein, human