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L Prakash

Publications and source records attributed to L Prakash.

At least 145 records · Page 8Linked to original sources
PubMed1985

Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase.

We have cloned CDC9, the structural gene for Saccharomyces cerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation. The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of start but before S phase. This high level of CDC9 mRNA then decays with an apparent half-life of ca. 20 min and remains at a low basal level throughout the rest of the cell cycle. The accumulation of CDC9 mRNA in late G1 is dependent upon the completion of start but not the CDC7 and CDC8 functions. Exposure of cells to UV light elicits an eightfold increase in DNA ligase mRNA levels.

Cell Cycle↗
PubMed1984

Isolation and characterization of the RAD2 gene of Saccharomyces cerevisiae.

We have cloned the RAD2 gene of Saccharomyces cerevisiae and used it to determine the size and direction of its transcript and to make rad2 deletion mutants. The RAD2 gene encodes a 3.3-kb transcript and the direction of transcription is leftwards, from EcoRI towards Bg/II. Deletions of the RAD2 gene have no effect on viability of vegetative cells or spores, or on sporulation.

Chromosome Deletion↗
PubMed1983

Molecular cloning and characterization of the RAD1 gene of Saccharomyces cerevisiae.

We have cloned the RAD1 gene of Saccharomyces cerevisiae and physically mapped it to a 4.0-kb DNA fragment from chromosome XVI. The RAD1 gene determines a transcript of 3.1 kb, and the direction of transcription was found to be leftwards, from EcoRI towards Bg/II (Fig. 1). Deletions of the RAD1 gene were made and were found to have no effect on viability of vegetative cells or spores, or on sporulation.

Chromosome Mapping↗
PubMed1983

Isolation and characterization of the RAD3 gene of Saccharomyces cerevisiae and inviability of rad3 deletion mutants.

The RAD3 gene of Saccharomyces cerevisiae is required for nicking of DNA containing pyrimidine dimers or interstrand crosslinks. We have cloned the RAD3 gene and physically mapped it to 2.6 kilobase of DNA. A DNA segment of the cloned RAD3 insert was ligated into plasmid YIp5, which transforms yeast by homologous integration, and shown to integrate at the RAD3 site in chromosome V, thus verifying the cloned DNA segment to be the RAD3 gene and not a suppressor. The RAD3 gene encodes a 2.5-kilobase mRNA, extending between the Kpn I site and the Sau3A1/BamHI fusion junction in plasmid pSP10, and the direction of transcription has been determined. The 2.5-kilobase transcript could encode a protein of about 90,000 daltons. We also show the deletions of the RAD3 gene to be recessive lethals, indicating that the RAD3 gene plays an important role in other cellular processes in addition to incision of damaged DNA.

Journal Article↗
PubMed1982

Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae.

Excision of pyrimidine dimers and interstrand DNA crosslinks was examined in the deletion mutants rad7-delta 1, rad23-delta 1, and rad7-delta 1 rad23-delta 1. These mutants remove pyrimidine dimers and crosslinks much less efficiently than the RAD+ strains; only 30-60% of pyrimidine dimers and 25-40% of crosslinks are removed even after prolonged incubation. The rad7 and rad23 mutations may represent defects in protein factors which increase the efficiency of the nicking enzyme complex or make chromatin more accessible to the nicking activity.

Biological Evolution↗
PubMed1982

Role of DNA repair in ethyl methanesulfonate-induced mutagenesis in Saccharomyces cerevisiae.

Ethyl methanesulfonate (EMS)-induced reversion of two sites which revert preferentially by GC to AT transitions, cycl-131 and cycl-115, has been examined in readiation sensitive, rad, mutants of yeast belonging to the rad52 epistasis group. The rad50, rad51, rad52, rad54 and rad56 mutants showed reducted reversion of both tester sites when stationary phase diploid cells were treated with EMS. No correlation was found between EMS-induced reversion and EMS-induced homologous mitotic intragenic recombination. Survival of rad6 rad52 double mutants following EMS treatment indicates that there is one epistasis group for the repair of EMS-induced lethal damage in yeast. A model involving misrepair mutagenesis of specific lesions is proposed to account for the experimental results.

DNA Repair↗
PubMed1982

Genetic control of excision of Saccharomyces cerevisiae interstrand DNA cross-links induced by psoralen plus near-UV light.

Excision of interstrand DNA cross-links induced by 4,5',8-trimethyl psoralen plus 360-nm light was examined in wild type (RAD+) and various radiation-sensitive (rad) mutants of Saccharomyces cerevisiae known to be defective in the excision of UV light-induced pyrimidine dimers. Alkaline sucrose sedimentation of DNA after incubation of psoralen-plus-light-treated cells indicated little or no nicking of cross-linked DNA in rad1-2, rad2-5, rad3-2, rad4-4, rad10-2, and mms19-1 mutants. In the rad14-2 mutant, substantial nicking was observed but to a much lesser extent than in the RAD+ strains, whereas the rad16-1 mutant was as proficient in nicking as the RAD+ strain. Removal of cross-links was also examined in RAD+, rad3-2, and rad14-2 strains by determining the sensitivity of alkali-denatured and -neutralized DNA to hydrolysis by S1 nuclease. No cross-link removal was observed in the rad3-2 mutants, and the rad14-2 mutant was much less efficient than the RAD+ strain in removing cross-links.

DNA Repair↗
PubMed1981

Purification and characterization of a uracil-DNA glycosylase from the yeast. Saccharomyces cerevisiae.

An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharomyces cerevisiae. The enzyme is active on both native and denatured PBS1 DNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA. The enzyme has a negative molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gel filtration. Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.

DNA Glycosylases↗
PubMed1981

Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: evidence for multiple functions of the RAD6 gene.

The rad6-1 and rad6-3 mutants are highly UV sensitive and show an increase in spontaneous and UV induced mitotic heteroallelic recombination in diploids. Both rad6 mutants are proficient in spontaneous and UV induced unequal sister chromatid recombination in the reiterated ribosomal DNA sequence and are deficient in UV induced mutagenesis. In contrast to the above effects where both mutants appear similar, rad6-1 mutants are deficient in sporulation and meiotic recombination whereas rad6-3 mutants are proficient. The differential effects of these mutations indicate that the RAD6 gene is multifunctional. The possible role of the RAD6 gene in error prone excision repair of UV damage during the G1 phase of the cell cycle in addition to its role in postreplication repair is discussed.

Alleles↗
PubMed1981

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations.

Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (rad1 rho 0). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m2, the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.

Cycloheximide↗
PubMed1981

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

Epistasis, Genetic↗
PubMed1981

Spontaneous mitotic recombination in mms8-1, an allele of the CDC9 gene of Saccharomyces cerevisiae.

The methyl methane sulfonate (MMS)-sensitive mutation mms8-1 increases the rate of spontaneous mitotic intragenic recombination at five heteroallelic loci on three chromosomes. Complementation, segregation, and mapping studies indicate that mms8-1 is allelic to cdc9, known to be defective in deoxyribonucleic acid ligase. Both mms8-1 and cdc9 mutants are lethal in combination with the recombination-defective mutant rad52-1. Genetic analysis of spontaneous red/white sectors in an ade2-1/ade2-1 ade5/+ mms8-1/mms8-1 strain shows nonreciprocal recombinational events involving long chromosome segments. We also observe greater than expected rates of simultaneous recombination at loci on different chromosomes in both wild-type and mms8-1 mutants.

Alleles↗
PubMed1981

Incision and postincision steps of pyrimidine dimer removal in excision-defective mutants of Saccharomyces cerevisiae.

cdc9, a temperature-sensitive mutant defective in polynucleotide deoxyribonucleic acid (DNA) ligase activity, accumulates low-molecular-weight DNA fragments (as measured by sedimentation of DNA in alkaline sucrose gradients) at the nonpermissive temperature after irradiation with ultraviolet light. This phenotype of cdc9 is a sensitive indicator of successful incision during excision repair of dimers. In strains containing excision-defective mutations in any of nine genes in combination with the cdc9 mutation, the absence of low-molecular-weight DNA at the nonpermissive temperature after ultraviolet treatment suggests that these mutants are incision defective, whereas the presence of low-molecular-weight DNA indicates that the mutants are defective in a step after incision. With rad1, rad2, rad3, rad4, and rad10 mutants, the molecular weight of the DNA remained unchanged after ultraviolet irradiation and incubation at the restrictive temperature, despite the presence of the cdc9 mutation; these mutants are therefore incision defective. Low-molecular-weight DNA was observed in rad14 cdc9 and rad16 cdc9 strains. With the rad16 strain, the accumulation of low-molecular-weight DNA correlated with the amount of excision taking place, whereas in the rad14 mutant strain, no evidence of dimer removal was obtained. Therefore, rad14 is likely to be defective in a step after incision.

DNA Repair↗
PubMed1979

Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae.

A DNA replication mutant of yeast, cdc8, was found to decrease UV-induced reversion of lys2-1, arg4-17, tyr1 and ura1. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following UV irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since UV-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.

Alleles↗
PubMed1979

Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers from the nuclear DNA of yeast was examined in two radiation-sensitive (rad) mutants and one methyl methanesulfonate-sensitive (mms) mutant of the yeast Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by an endonuclease activity prepared from crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad7, rad14 and mms19 mutants were found to be defective in their ability to remove UV-induced dimers from nuclear DNA. All three mutants belong to the same epistatic group as the other mutants involved in excision-repair. All three mutants show enhanced UV-induced mutations. The rad14 mutant also shows epistatic interactions with genes in the other two UV repair pathways.

DNA Repair↗
PubMed1977

Defective thymine dimer excision in radiation-sensitive mutants rad10 and rad16 of Saccharomyces cerevisiae.

Two rad mutants of yeast, rad10 and rad16, are shown to be defective in the removal of UV-induced pyrimidine dimers since DNAs obtained from irradiated cells following a post-irradiation incubation in the dark still retain UV-endonuclease-sensitive sites. Both rad10 and rad16 mutants are in the same pathway of excision-repair as the rad1, rad2, rad3 and rad4 mutants.

Crosses, Genetic↗