Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae.

Abstract

The RADI gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RADI gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions + 1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RADI gene, which also deletes the +1 ATG and 11 additional codons in the RADi open reading frame, partially complements UV sensitivity of a rad1A mutant, we examined the role of the + 1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the + 1 ATG codon to ATC affected the complementation ability of the RADI gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RADI function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RADI-lacZ fusions showed that the RADI open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360. The RAD1 gene of Saccharomyces cerevisiae is one of 10 genes, RADI, RAD2, RAD3, RAD4, RAD7, RADIO, RAD14, RAD16, RAD23, and MMS19, involved in excision repair of DNA damaged with UV light, bulky adducts, or cross-linking agents (17-19, 30, 39). Of these, the RADI, RAD2, RAD3, RAD4, RADIO, and MMSJ9 genes seem to be required for incision of damaged DNA since their mutants are highly defective in this process, whereas the remaining four genes affect incision only partially (17,18,30,39). To analyze the individual and the combined role of proteins encoded by these incision genes, we have cloned and characterized several of them (10-12, 16, 20, 24, 25, 27, 28). Some of these genes have also been cloned and studied by others (21-23, 38, 40). The cloned genes are being used to overproduce their proteins to facilitate their purification, characterization, and assembly of the incision complex in vitro. Such an approach has allowed successful in vitro assembly and characterization of the UVRABC incision complex in Escherichia coli (34).As an important step toward achieving the above goals, we have examined the primary structure of the proteins encoded by the RAD2, RAD3, RAD7, and RADIO genes, as deduced from their nucleotide sequence (16,24,27,28). In the RAD3-encoded protein sequence we identified a segment which is homologous with a consensus sequence present in a large number of proteins which bind and hydrolyze ATP, suggesting that RAD3 protein also possesses a similar activity (27). The RADIO protein sequence indicates the presence of putative DNA-binding regions (28). Interestingly, significant amino acid sequence homology has been observed between the yeast RAD10 protein and the protein encoded by the human excision gene ERCC-J, suggesting that repair proteins in eucaryotes are conserved (37).In this paper, we present the nucleotide and the deduced amino acid sequences of RADI. The RADI open reading frame encodes a protein of 1,100 amino acids with a molec...

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

“…Three of the radl mutant alleles examined in this study were generated by oligonucleotide-directed mutagenesis and included mutation of the ATG (methionine) codon at position +1 in the RADI open reading frame to ATC (isoleucine) in plasmid pRR113, mutation of the ATG codon at position +334 in the RADI open reading frame to ATC in plasmid pRR127, and mutation of both ATG codons at positions +1 and +334 to ATC in plasmid pRR129 (26 Reynolds, unpublished observations). The +1 ATC mutation affects the DNA repair function of RADI; radlA cells bearing this mutant radl allele on plasmid pRR113 are UV sensitive, but the UV sensitivity of these cells is intermediate between that of the RADW and the radlA cells (26). The residual level of UV resistance provided by the +1 ATC mutant radl allele probably derives from low-level translational readthrough from the +1 AUC codon in the radl mRNA.…”

“…Strain RS69-8B was transformed with the following plasmids carrying the RADI gene or various radl mutant alleles; pRR117 (RADI +), pRR113 (+1 ATC), pRR127 (+334 ATC), pRR129 (+1 ATC, +334 ATC), and pRR125 (which carried the radl gene deleted for 2.7 kb of 5' DNA including the first 12 RADI codons). These plasmids have been previously described (26).…”

“…radi mutants. We used radl mutants in which the + 1 ATG or the +334 ATG or both ATGs in the coding sequence were mutated to ATC by oligonucleotide-directed mutagenesis and carried on a single-copy CEN plasmid, YCp5O (26). In strain RS69-8B (Table 1) containing the HIS3::pRS6 recombination system, the RADI and RAD52 genes were replaced by URA3 and TRPI, respectively, and a ura3 mutant was selected on 5-fluoroorotic acid medium (3).…”

“…Reynolds) and used to replace the genomic RAD1 gene from positions -212 to +3853 (26) by the URA3 gene. In plasmid pRR27 the HindIII fragment of the RADI gene from positions -212 to +3853 was removed and replaced by the 1.1-kb HindIII URA3 fragment from plasmid YEp24.…”

“…In plasmid pRR27 the HindIII fragment of the RADI gene from positions -212 to +3853 was removed and replaced by the 1.1-kb HindIII URA3 fragment from plasmid YEp24. For replacing the genomic RAD1 gene from positions -212 to +3853 by the LEU2 gene, a 7-kb BamHI-BamHI DNA fragment containing the LEU2 gene and flanked by the 5' and 3' RADI sequences was obtained from plasmid pRR46 (26) and used to transform yeast strains to Leu+. The genomic RAD2 gene from positions +129 to +2911 (15) firmed by complementation analysis with known rad mutations.…”