title: Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus.
abstract: Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2-3 times higher than that of udgB at 70, 74, and 78 degrees C when it was monitored by beta-glucosidase reporter assay. We developed hisD(3110), hisD(3113), hisD(3115), and hisD(174) marker allele that can specifically detect G:C-->A:T, C:G-->A:T, T:A-->A:T, and A:T-->G:C base-substitutions, respectively, by His(+) reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His(+) reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C-->A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees C culture, increased ratio of the mutation rate compared with the udg(+) strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G-->A:T, T:A-->A:T, and A:T-->G:C between udgA,B double mutant and the parent udg(+) strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C-->A:T transition mutations.
authors.affiliation: School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Sakai
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Tokishita
authors.forename: Kayo
authors.initials: K
authors.lastname: Mochizuki
authors.forename: Ayako
authors.initials: A
authors.lastname: Motomiya
authors.forename: Hideo
authors.initials: H
authors.lastname: Yamagata
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Ohta
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D059645:Mutation Rate; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D051981:Uracil-DNA Glycosidase