Dual Role for the Yeast THI4 gene in Thiamine Biosynthesis and DNA Damage Tolerance Carlos R. Machado 1 , Uta M. Praekelt 1,2 , Regina Costa de Oliveira 1 Ana Carolina C. Barbosa 1 , Kerry L. Byrne 2 , Peter A. Meacock 2 and Carlos F. M. Menck 1,3 * 1 Depto. de Biologia, Instituto de Biocie Ãncias, Universidade de Sa Äo Paulo, CP 11461 Sa Äo Paulo, 05422-970, Brazil 2 Dept. of Genetics, University of Leicester, University Road Leicester LE1 7RH, UK 3 Depto. de Microbiologia Instituto de Cie Ãncias Biome Âdicas, Universidade de Sa Äo Paulo, Av. Prof. Lineu Prestes 1374, Sa Äo Paulo 05508-900, SP, Brazil The THI4 gene of Saccharomyces cerevisiae encodes an enzyme of the thia- mine biosynthetic pathway. The plant homolog thi1, from Arabidopsis thaliana, is also involved in thiamine biosynthesis; but was originally cloned due to its capacity to complement DNA repair de®cient pheno- types in Escherichia coli. Here, the behavior of a thi4 disrupted strain was examined for increased sensitivity to treatment with the DNA damaging agents ultraviolet radiation (UV, 254 nm) and methyl methanesulfonate (MMS). Although the thi4 null mutant showed a similar level of survival as the wild-type strain, a higher frequency of respiratory mutants was induced by the two treatments. A similar phenotype was seen with wild- type strains expressing an antisense THI4 construct. Further analysis of respiratory mutants revealed that these were due to mutations of mito- chondrial DNA (mtDNA) rather than nuclear DNA, consisting of r  petite mutants. Moreover, the frequency of mutations was unaffected by the presence or absence of thiamine in the growth medium, and the defect leading to induction of petites in the thi4 mutant was corrected by expression of the Arabidopsis thi1 gene. Thus, Thi4 and its plant homolog appear to be dual functional proteins with roles in thiamine biosynthesis and mitochondrial DNA damage tolerance. # 1997 Academic Press Limited Keywords: mtDNA stability; thiamine biosynthesis; THI4; DNA damage tolerance; plant DNA repair *Corresponding author Introduction The integrity of genetic material is continuously threatened by potentially harmful by-products of normal metabolism and by environmental agents such as radiation and toxic chemicals. Organisms have a complex array of DNA repair enzymes that are able to recognize and remove, or simply toler- ate the damaged regions of their genomes. Such enzymes have been particularly well characterized in Escherichia coli, and yeast has served as a useful model for the study of eukaryotic DNA repair (reviewed by Hoeijmakers, 1993). Studies of eukaryotic DNA have mainly focused on the repair of the nuclear genome, and until recently it was widely assumed that repair of organellar DNA does not occur, nor that it is necessary because of the redundancy of genetic information in the mul- tiple copies of organellar DNA. This view was sup- ported by the observation that mtDNA is subject to a much higher mutation rate than nuclear DNA (Richter et al., 1988) and that some bulky lesions, such as cyclobutane pyrimidine dimers, persist in mtDNA of mammals (LeDoux et al., 1992) and in mitochondria and chloroplasts of plants (Chen et al., 1996). However, repair enzymes, such as UV endonuclease, have been demonstrated in mamma- lian (Tomkinson et al., 1990) and plant organelles (Benson & Warner, 1987) suggesting that some degree of repair occurs in these ones. The discovery of a homolog of the E. coli RecA protein in plant chloroplasts (Cerutti et al., 1992) may indicate that even irreversibly damaged orga- nellar genomes could still be replicated with high ®delity employing undamaged regions in extra copies of templates to ®ll daughter strand gaps. The importance of recombination in the mainten- Abbreviations used: mtDNA, mitochondrial DNA; MMS, methyl methanesulfonate; w.t., wild-type; TTC, triphenyltetrazolium chloride; DAPI, 4 0 ,6-diamidino-2- phenylindole. J. Mol. Biol. (1997) 273, 114±121 0022±2836/97/410114±08 $25.00/0/mb971302 # 1997 Academic Press Limited