CLONING OF SCHIZOSACCHAROMYCES-POMBE RPH16(-CEREVISIAE RAD16 GENE(), A GENE HOMOLOGOUS TO THE SACCHAROMYCES)

The RAD16 gene is involved in the nucleotide excision repair of UV dam age in the transcriptional silenced mating type loci (Terleth et al., 1990 and Bang et al., 1992) and in non-transcribed strands of active g enes in Saccharomyces cerevisiae (Verhage et al., 1994). Using touchdo wn-PCR with primers derived from various domains of the S. cerevisiae Rad16 protein, a specific Schizosaccharomyces pombe probe was isolated . This probe was used to obtain the complete RAD16 homologous gene fro m a S. pombe chromosomal bank. DNA sequence analysis of the rph16(+) g ene revealed an open reading frame of 854 amino acids. Comparison of t he amino acid sequences of the Rhp16 and Rad16 proteins showed a high level of conservation: 68% similarity. The Rhp16 protein sequence cont ains the two Zn-finger motifs and the putative helicase domains as fou nd in the Rad16 protein. Like the RAD16, the rph16(+) gene is UV-induc ible (Bang et al., 1995). Tn analogy with the radl6 mutant, the rhpl6 disruption mutant is viable and grows normally, indicating that the ge ne does not have an essential function. The rhpI6 disruption mutant is not sensitive for UV but is sensitive for cisplatin. The rhp16(+) gen e cloned behind the GAL1 promoter partially complements the UV sensiti vity and the defect in the non-transcribed strand DNA repair of a S. c erevisiae rad16 mutant, indicating functional homology between the rhp 16(+) and RAD16 genes. The structural and functional homology between the two genes suggests that the RAD16 dependent subpathway of NER for the repair of non-transcribed DNA is evolutionary conserved.