The nucleotide sequence of the<i>RAD3</i>gene of<i>Saccharomyces cerevisiae</i>: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region

Reynolds, Paul; Higgins, David R.; Prakash, Louise; Prakash, Satya

doi:10.1093/nar/13.7.2357

Cited by 84 publications

(39 citation statements)

References 60 publications

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“…In xlgv7, the A motif is located at amino acid residues 120-129. This sequence, Leu (n)6 Gly Lys Thr, is very similar to the sequences found on the yeast RAD 18 and RAD3 proteins (Reynolds et al 1985;Jones et al 1988) and several bacterial proteins such as RecA and RecB, which bind and hydrolyze ATP (see Table 2). The B motif is located 196 amino acids away from the A motif (315-327 see Table 2) and consists of Lys n n nPro n n n Leu Leu Tyr Lys Asp.…”

Section: Cloning and Characterization Of The Xlgv7 Cdnasupporting

confidence: 66%

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

Miller¹,

Kloc²,

Reddy³

et al. 1989

Genes Dev.

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The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system {CNS) of tadpoles and adult frogs. This cDNA {xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal {Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine {His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cellspecific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15--16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.

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Section: Cloning and Characterization Of The Xlgv7 Cdnasupporting

confidence: 66%

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

Miller¹,

Kloc²,

Reddy³

et al. 1989

Genes Dev.

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“…Small-scale plasmid preparations from E. coli were made by a modification of the boiling method (9). S. cerevisiae was transformed by treating intact cells within lithium acetate to promote DNA uptake (11 (25). In the experiment shown in Table 8 Deletion of RAD genes.…”

Section: Methodsmentioning

confidence: 99%

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

1988

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The RADI gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RADI gene on genetic recombination.Mitotic intrachromosomal and interchromosomal recombination in RAD+, radi, rad52, and other rad mutant strains was examined. The radl deletion mutation and some radl point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the radl rad52 double mutant, indicating that the RADI and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RADI gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the radlA mutation did lower the frequency of integration of lnear plasmids and DNA fragments into homologous genomic sequences. We suggest that RADI plays a role in recombination after the formation of the recombinogenic substrate.In the yeast Saccharomyces cerevisiae, 10 genes, RAD1, RAD2, RAD3, RAD4, RAD7, RADIO, RAD14, RAD16, RAD23, and MMS19, are known to be involved in excision repair of DNA damaged by UV light and by other agents that deform the DNA helix. The radi, rad2, rad3, rad4, and radiO mutants are highly sensitive to UV light and to other DNA-damaging agents and are totally defective in the incision of damaged DNA (18,27,36). The mms19, rad7, radI4, radi6, and rad23 mutants are not as sensitive to DNAdamaging agents and differ in excision proficiency from each other (18,19). In their absolute requirement for incision, the RADI, RAD2, RAD3, RAD4, and RADIO genes of S. cerevisiae resemble the Escherichia coli uvrA, uvrB, and uvrC genes. The UvrABC enzyme complex of E. coli cuts the damaged strand at two sites, which are 7 to 8 nucleotides 5' and 3 to 4 nucleotides 3' from the damage (29, 37). The UvrD and Poll enzymes catalyze the release of the UvrABC enzyme complex from the damaged site (4, 10).In contrast to the E. coli uvrA, uvrB, and uvrC genes, which are required only for incision of damaged DNA, some of the yeast incision genes seem to be involved in other cellular processes as well. The RAD3 gene, in addition to its role in incision, is essential for cell viability; rad3 deletion mutants are inviable (8, 21), and various rad3 mutants that are defective only in the excision repair function or that are temperature sensitive for growth but proficient in DNA repair exist (22, 23; R. Schiestl and S. Prakash, unpublished observations). The RAD3 protein has been purified in our lab...

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“…The precise location of each mutation was determined by DNA sequencing of the relevant DNA fragments (19 - Fig. 2 has been noted by others (29). A second region of interest is located near the carboxy terminus of the Rad3 protein sequence between amino acids 592 and 611.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

Naumovski

Friedberg

1986

Mol. Cell. Biol.

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The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylaniine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.RAD3 is one of at least five genes in Saccharomyces cerevisiae that are necessary for the excision repair of base damage caused by UV radiation or by many mutagenic chemicals (30,37). Of these, the RADI, RAD2, RAD4, and RADIO genes are not required for the growth and viability of haploid yeast cells (22,36,38; R. Fleer, G. Pure, and E. C. Friedberg, unpublished observations). In contrast, the RAD3 gene is essential, and disruption of the gene renders haploid yeast cells inviable (10,21). The nature of the essential function of the RAD3 gene is not known; however, the observation that some rad3 mutants defective in the incision of damaged DNA are viable and apparently grow normally led us to suggest that the essential function may be distinct from that required for excision repair (21). In an effort to learn more about the organization of the RAD3 gene with respect to its two known functions, we examined the effect of mutations at two distinct regions by using oligonucleotide-directed mutagenesis. The selection of these sites was based on the identification of regions in which the predicted amino acid sequence shows homology with that of other sequenced proteins or genes. One subset of these proteins consists of known purine ribonucl...

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The nucleotide sequence of theRAD3gene ofSaccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region

Cited by 84 publications

References 60 publications

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis.

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

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

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