Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae

Meiotic recombination in yeast is associated with heteroduplex formation. Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected. Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation. Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast. In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene. These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients).     

The mutator form of polymerase beta with amino acid substitution at tyrosine 265 in the hinge region displays an increase in both base substitution and frame shift errors

This study describes the first complete in vitro error specificity analysis of a mutator DNA polymerase that is altered in a residue not predicted to contact either the DNA or dNTP substrate. We examined this mutator form of polymerase beta (Y265C) in order to elucidate the critical role tyrosine 265 plays in the accuracy of DNA synthesis. Our results demonstrate that an increase in both frame shift errors in homonucleotide repeat sequences and base substitution errors contribute nearly equally to the Y265C mutator phenotype. The models described for production of these errors, primer/template misalignment and base misincorporation, respectively, are distinctly different, suggesting the Y265C alteration affects discrimination against both types of error production pathways. In addition, Y265C displays a 530-fold increase in multiple errors within the 203-base pair target region examined, relative to that of wild type. Processivity studies revealed that Y265C retains the near distributive nature of DNA synthesis characteristic of the wild type polymerase beta. Therefore, multiple errors exhibited by Y265C most likely result from independent polymerase binding events. Localization of tyrosine 265 in the X-ray crystallographic structure suggests this residue may play a role in mediating a conformational change of the polymerase [Pelletier, H., et al. (1996) Biochemistry 35, 12742-12761]. A conformational change is predicted to enhance the accuracy of DNA synthesis by imposing an induced fit selection against premutational intermediates. The observed loss of discrimination against both misalignment-mediated and misincorporation-mediated errors produced by polymerase Y265C is consistent with such a model.     

Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae

We recently showed the involvement of the L-arginine/nitric oxide (NO) pathway in acid-induced duodenal mucosal bicarbonate secretion in rats. The aim of the present study was to confirm this observation in pigs by direct measurements of NO production. Experiments were performed on 16 anaesthetized pigs of both sexes treated with guanethidine (6 mg kg-1, intravenously). A duodenal segment, devoid of pancreaticobiliary influxes, was perfused with saline and the duodenal mucosal bicarbonate secretion was calculated from continuous measurements of pH and PCO2. The perfusate contents of NO and its oxidative product nitrite were determined by chemiluminescence, after reduction of nitrite to NO. Luminal acidification with 30 mM hydrochloric acid increased the output of bicarbonate as well as NO to the perfusate, by 195 +/- 45% and 106 +/- 10%, respectively. These responses to acid were markedly inhibited by adding the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.3 mM) to the perfusate. The inhibitory effect of L-NMMA could be reversed by administration of L-arginine (3 mM). The study presents simultaneous measurements of bicarbonate and NO outputs to a duodenal luminal perfusate. The results strongly support the view that the L-arginine/NO pathway is involved in the acid-induced duodenal mucosal bicarbonate secretory response.     

The influence of mutation rad57-1 on the fidelity of DNA double-strand gap repair in Saccharomyces cerevisiae

The role of the RAD57 gene in double-strand gap (DSG) repair has been examined. The repair of a linearized plasmid, bearing a DSG, has been analyzed in a rad57-1 mutant of Saccharomyces cerevisiae. For effective rejoining of the ends of plasmid DNA in the rad57 mutant the sequence of chromosomal DNA homologous to the DSG region is required. However, DSG repair (restoration of plasmid circularity) in rad57 cells is not accompanied by the recovery of DSGs. The DSG repair, which depends on an homologous chromosomal DNA sequence, requires the cohesive ends of DSGs. The non-cohesive-ended DSGs are repaired in rad57 cells by a pathway independent of the homologous recombination between chromosomal and plasmid DNA. We presume that the rad57-1 mutation is connected with the inhibition of DNA repair synthesis, required for filling the DSG. This situation produces a condition of "homology-dependent ligation", the alternative minor mechanism of recombinational DSG repair, that takes place in mutant cells. A molecular model for "homology-dependent ligation" in rad57 cells is proposed.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase alpha and is required for cell cycle progression in G2/M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cdelta) shows slow growth at low temperature (15 degrees C), while at 30 degrees C and 37 degrees C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Pol1, the catalytic subunit of DNA polymerase alpha. The pol1-1 /yjr043cdelta double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cdelta. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cdelta mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication.     

a/alpha-control of DNA repair in the yeast Saccharomyces cerevisiae: genetic and physiological aspects

It has long been known that diploid strains of yeast are more resistant to gamma-rays than haploid cells, and that this is in part due to heterozygosity at the mating type (MAT) locus. It is shown here that the genetic control exerted by the MAT genes on DNA repair involves the a1 and alpha 2 genes, in a RME1-independent way. In rad18 diploids, affected in the error-prone repair, the a/alpha effects are of a very large amplitude, after both UV and gamma-rays, and also depends on a1 and alpha 2. The coexpression of a and alpha in rad18 haploids suppresses the sensitivity of a subpopulation corresponding to the G2 phase cells. Related to this, the coexpression of a and alpha in RAD+ haploids depresses UV-induced mutagenesis in G2 cells. For srs2 null diploids, also affected in the error-prone repair pathway, we show that their G1 UV sensitivity, likely due to lethal recombination events, is partly suppressed by MAT homozygosity. Taken together, these results led to the proposal that a1-alpha 2 promotes a channeling of some DNA structures from the mutagenic into the recombinational repair process.     

Role of the RAD57 gene in the repair of double-stranded DNA gaps in the yeast Saccharomyces cerevisiae

The linearized plasmid with complementary (cohesive) ends was shown to restore the circular form in cells of the rad57 mutant with a lower efficiency than in Rad+ cells. This process proved to be cold-sensitive in mutant cells, in contrast to wild-type cells. When mutant cells were shifted from 23 up to 36 degrees C, the repair efficiency increased approximately 1.5 times. In most cases examined, the repair was not accompanied by the doublestrand gap repair within the break site and did not depend on temperature. Homology between chromosomal and plasmid DNA sequences in the break region and the presence of cohesive ends were shown to be essential for the repair of linearized plasmids with a double-strand gap in cells of the rad57 mutant. Degradation of cohesive ends of the linearized plasmid during its repair in rad57 cells is insignificant. Possible mechanisms of linearized plasmid repair in the rad57 mutant are proposed.     

Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage

A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation. The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein. These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair.     

The absence of strand-specific repair for the DNA polymerase pol gene in Deinococcus radiodurans

Deinococcus radiodurans is characterized by its extreme resistance to ionizing radiation. Although Mn+2 was taken up by the cells at a high speed, the absorption of Mn+2 by the cells did not affect their survival rate when irradiated by UV light. However, the Mn-induced-cell-division (Mn-CD) cells do lose their resistance to UV radiation. This can be explained by the fact that the Mn-CD cells were less efficient to remove pyrimidine dimers on damaged DNA. We also found that there is no strand-specific repair of the pol gene in this organism.     

Partial purification of Pde1 from Saccharomyces cerevisiae: enzymatic redundancy for the repair of 3'-terminal DNA lesions and abasic sites in yeast

Earlier work indicates that the major DNA repair phosphodiesterase (PDE) in yeast cells is the well-characterized Apn1 protein. Apn1 demonstrates both Mg2+-independent PDE activity and Mg2+-independent class II apurinic/apyrimidinic (AP) endonuclease activity and represents greater than 90% of the activity detected in crude extracts from wild-type yeast cells. Apn1 is related to Echerichia coli endonuclease IV, both in its enzymatic properties and its amino acid sequence. In this work, we report the partial purification of a novel yeast protein, Pde1, present in Apn1-deficient cells. Pde1 is purified by sequential BioRex-70, PBE118, and MonoS chromatography steps using a sensitive and highly specific 3'-phosphoglycolate-terminated oligonucleotide-based assay as a measure of PDE activity. Mg2+-stimulated PDE and Mg2+-stimulated class II AP endonuclease copurify during this procedure. These results indicate that yeast, like many other organisms studied to date, has enzymatic redundancy for the repair of 3'-blocking groups and abasic sites.     

Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.     

Substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae: excision of guanine lesions produced in DNA by ionizing radiation- or hydrogen peroxide/metal ion-generated free radicals

We have investigated the substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae (yOgg1 protein) for excision of modified DNA bases from oxidatively damaged DNA substrates using gas chromatography/isotope dilution mass spectrometry. Four DNA substrates prepared by treatment with H2O2/Fe(III)-EDTA/ascorbic acid, H2O2/Cu(II) and gamma-irradiation under N2O or air were used. The results showed that 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were efficiently excised from DNA exposed to ionizing radiation in the presence of N2O or air. On the other hand, 8-OH-Gua and FapyGua were not excised from H2O2/Fe(III)-EDTA/ascorbic acid-treated and H2O2/Cu(II)-treated DNA respectively. Fourteen other lesions, including the adenine lesions 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine, were not excised from any of the DNA substrates. Kinetics of excision significantly depended on the nature of the damaged DNA substrates. The findings suggest that, in addition to 8-OH-Gua, FapyGua may also be a primary substrate of yOgg1 in cells. The results also show significant differences between the substrate specificities of yOgg1 protein and its functional analog Fpg protein in Escherichia coli.     

Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae: requirement of replication fork blocking (Fob1) protein and the role of RNA polymerase I

Saccharomyces cerevisiae carries approximately 150 copies of rDNA in tandem repeats. It was found that the absence of an essential subunit of RNA polymerase I (Pol I) in rpa135 deletion mutants triggers a gradual decrease in rDNA repeat number to about one-half the normal level. Reintroduction of the missing RPA135 gene induced a gradual increase in repeat number back to the normal level. Gene FOB1 was shown to be essential for both the decrease and increase of rDNA repeats. FOB1 was shown previously to be required for replication fork blocking (RFB) activity at RFB site in rDNA and for recombination hot-spot (HOT1) activity. Thus, DNA replication fork blockage appears to stimulate recombination and play an essential role in rDNA expansion/contraction and sequence homogenization, and possibly, in the instability of repeated sequences in general. RNA Pol I, on the other hand, appears to control repeat numbers, perhaps by stabilizing rDNA with the normal repeat numbers as a stable nucleolar structure.     

The prevalence of herpes family virus DNA in the conjunctiva of patients positive and negative for human immunodeficiency virus using the polymerase chain reaction

OBJECTIVE: To help understand the pathogenesis of herpes family virus ocular infection among patients positive for HIV, the authors compared the rates of detection of herpes family virus DNA from the conjunctiva of patients who are positive and negative for human immunodeficiency virus (HIV) using the polymerase chain reaction (PCR). DESIGN: Cross-sectional study. PARTICIPANTS: The conjunctival scrapings of 30 patients positive for HIV and 30 patients negative for HIV were examined. INTERVENTION: PCR was used to assay for the presence of herpes simplex virus type 1 (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) DNA (n = 240 samples). MAIN OUTCOME MEASURE: The rate of detection of virus DNA in the two groups, controlling for age, gender, and race, was measured. RESULTS: HSV and VZV DNA were not detected in any of the HIV-positive or HIV-negative samples. CMV DNA was detected in 20% (6 of 30) of patients positive for HIV and was undetected in control subjects negative for HIV (P = 0.01). EBV DNA was detected in 40% (12 of 30) of patients positive for HIV and in 47% (14 of 30) of control subjects negative for HIV (P = 0.58). CONCLUSIONS: There was no difference in the frequency of detection of HSV, VZV, or EBV DNA from the conjunctiva of patients positive or negative for HIV. Only CMV DNA was detected at a significantly higher rate in the conjunctiva of patients positive for HIV compared with control subjects negative for HIV. These different rates of peripheral virus shedding may be one possible explanation for the different rates of clinical infection among the herpes family viruses among patients positive for HIV.     

The H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-supplemented medium accompanying the decrease in intracellular pH and cell viability

Saccharomyces cerevisiae plasma membrane H(+)-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi). The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.     

The Saccharomyces cerevisiae TSC10/YBR265w gene encoding 3-ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca2+-sensitive csg2Delta mutant

Saccharomyces cerevisiae csg2Delta mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2Delta mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Delta suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed in Escherichia coli and isolated by nickel-nitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine.