Cloning of Schizosaccharomyces pombe rph16+, a gene homologous to the Saccharomyces cerevisiae RAD16 gene.
The RAD16 gene is involved in the nucleotide excision repair of UV damage in the transcriptional silenced mating type loci (Terleth et al., 1990 and Bang et al., 1992) and in non-transcribed stands of active genes in Saccharomyces cerevisiae (Verhage et al., 1994). Using touchdown-PCR with primers derived from various domains of the S. cerevisiae Rad 16 protein, a specific Schizosaccharomyces pombe probe was isolated. This probe was used to obtain the complete RAD16 homologous gene from a S. pombe chromosomal bank. DNA sequence analysis of the rph16+ gene revealed an open reading frame of 854 amino acids. Comparison of the amino acid sequences of the Rhp16 and Rad16 proteins showed a high level of conservation: 68% similarity. The Rhp16 protein sequence contains the two Zn-finger motifs and the putative helicase domains as found in the Rad16 protein. Like the RAD16, the rph16+ gene is UV-inducible (Bang et al., 1995). In analogy with the rad16 mutant, the rhp16 disruption mutant is viable and grows normally, indicating that the gene does not have an essential function. The rhp16 disruption mutant is not sensitive for UV but is sensitive for cisplatin. The rhp16+ gene cloned behind the GAI 1 promoter partially complements the UV sensitivity and the defect in the non-transcribed strand DNA repair of a S. cerevisiae rad16 mutant, indicating functional homology between the rhp16+ 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.
Bang
D D
DD
Department of Biochemistry, Leiden Institute of Chemistry, Gorlaeus laboratories, Leiden University, The Netherlands.
Ketting
R
R
de Ruijter
M
M
Brandsma
J A
JA
Verhage
R A
RA
van de Putte
P
P
Brouwer
J
J
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology; D003001:Cloning, Molecular; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015345:Oligonucleotide Probes; C101945:RHP16 protein, S pombe; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D000251:Adenosine Triphosphatases; C075467:RAD16 protein, S cerevisiae; D002945:Cisplatin