The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control

Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase ζ, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed.     

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F₂ intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY × BUF)F₁× BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait. Received: 15 April 1997 / Accepted: 8 May 1998     

Locomotion is not a privilege after birth: Ultrasound images of viviparous shark embryos swimming from one uterus to the other

Underwater ultrasound, a new tool for observing the internal body parts of aquatic animals by scuba divers, allowed us long‐term and frequent observations of the embryos of captive aquatic vertebrates. New ultrasound data of captive tawny nurse sharks (Nebrius ferrugineus) revealed that their embryos frequently migrate between the right and left uteri during gestation. This report is the first reliable evidence of active embryonic locomotion in live‐bearing vertebrates and is contradictory to the concept of “sedentary embryo” which has mainly arisen from studies of mammals. The tawny nurse shark is unique among orectolobiform sharks, in which the embryo develops by feeding on sibling eggs in utero. Thus, we hypothesized that swimming aids in an efficient search and capture of these eggs in the uterine environment.     

Detection of a Soluble Form of CD109 in Serum of CD109 Transgenic and Tumor Xenografted Mice

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is expressed at high levels in some human tumors including squamous cell carcinomas. As CD109 is reportedly cleaved by furin and its soluble form is secreted into culture medium in vitro, we hypothesized that CD109 could serve as a tumor marker in vivo. In this study, we investigated CD109 as a novel serum tumor marker using transgenic mice that overexpress mouse CD109 (mCD109-TG mice) and tumor xenografted mice inoculated with human CD109 (hCD109)-overexpressing HEK293 cells. In sera and urine of mCD109-TG mice, mCD109 was detected using western blotting. In xenografted mice, hCD109 secreted from inoculated tumors was detected in sera, using western blotting and CD109 ELISA. Concentrations of tumor-secreted CD109 increased proportionally as tumors enlarged. Concentrations of secreted CD109 decreased notably by 17 h after tumor resection, and became undetectable 48 h after resection. The half-life of tumor-secreted CD109 was about 5.86±0.17 h. These results indicate that CD109 is present in serum as a soluble form, and suggest its potential as a novel tumor marker in patients with cancers that express CD109.     

Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7

Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase zeta that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.