Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.     

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.     

An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae

An allele of RFA1, the largest subunit of the single-stranded DNA-binding complex RP-A, was identified as a suppressor of decreased direct-repeat recombination in rad1 rad52 double mutants. In this study, we used two LEU2 direct-repeat assays to investigate the mechanism by which the rfa1-D228Y allele increases recombination. We found that both intrachromatid and sister chromatid recombination are stimulated in rfa1-D228Y strains. In a rad1 rad52 background, however, the majority of the increased recombination is caused by stimulation of deletion events by an intrachromatid recombination mechanism that is likely to be single-strand annealing. Studies in which an HO endonuclease cut was introduced between the two leu2 copies indicate that the rfa1-D228Y mutation partially suppresses the rad52 defect in recovering recombination products. Furthermore, molecular analysis of processing and product formation kinetics reveals that, in a rad52 background, the rfa1-D228Y mutation results in increased levels of recombinant products and the disappearance of large single-stranded intermediates characteristic of rad52 strains. On the basis of these results, we propose that in the absence of wild-type Rad52, the interaction of RP-A with single-stranded DNA inhibits strand annealing, and that this inhibition is overcome by the rfa1-D228Y mutation.     

Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae

Firing rate histogram is a widely used mathematical method for representing the activity of single neurons and small neural networks. Nevertheless, observation of fine temporal modulation or correlations of spike trains might be troublesome if the mean firing rate is low or rapid local changes occur. The spike density function (SDF) obtained by convolving the spike train with smooth and continuous kernel function proves to be a more appropriate approach in characterization of the firing pattern. The resulting time-function is a continuous and derivable one, thus it can be used as a dynamical variable of the neuronal activity. In the present paper applications of SDF in analysis of the firing patterns of Lymnaea neurons are described and its performance is compared to other quantitative methods.     

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.     

Cell cycle regulation of the Saccharomyces cerevisiae polo-like kinase cdc5p

Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G2/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.     

Induction and repair of double-strand breaks in the replicating DNA of HeLa cells

The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed.     

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells

Recently the concept of dissociative identity disorder (formerly known as multiple personality disorder) has attracted increasing public and scientific interest. However, it is rarely diagnosed in the clinical setting. the reported case of a 47-year-old woman with a history of child abuse demonstrates the problems of differential diagnosis. A number of psychopathologic symptoms pointed to a multiple personality disorder, but in the follow-up psychotic symptoms such as delusions, possible hallucinations and bizarre behavior clearly emerged. The differential diagnosis of dissociative identity disorder includes paranoid schizophrenia, as in the case described, borderline personality disorder, hysteria, simulation and the false memory syndrome. Finally, social and cultural factors have to be considered.     

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.     

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.     

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

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.     

Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae

An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination. The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats). While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length. The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome. An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.     

Physiology of Saccharomyces cerevisiae during cell cycle oscillations

Synchronized populations of Saccharomyces cerevisiae CBS 426 are characterized by autonomous oscillations of process variables. CO2 evolution rate, O2 uptake rate and heat production rate varied by a factor of 2 for a continuous culture grown at a dilution rate of 0.10 h-1. Elemental analysis showed that the carbon mass fraction of biomass did not change. Since the reactor is not at steady state, the elemental and energy balances were calculated on cumulated quantities, i.e. the integral of the reaction rates. It was possible to show that carbon, degree of reduction and energy balances matched. Application of simple mass balance principles for non-steady state systems indicated that oscillations were basically characterized by changes in biomass production rate. In addition, the amount of intermediates, e.g. ethanol or acetate, produced or consumed was negligible. Growth rate was low during the S-phase (0.075 h-1) and high during the G2, M and G1 phases (0.125 h-1) for a constant dilution rate of 0.10 h-1. However, nitrogen, ash, sulfur and potassium content showed systematic increases during the S-phase (bud initiation). Cell component analyses showed that changes in cellular fractions during oscillations (storage carbohydrate content decreased during the S-phase) were due to changes in production rates, particularly for protein and carbohydrates. Nevertheless, using the data evaluation techniques for dynamic systems presented here, it was shown that storage carbohydrates are not consumed during the S-phase. Only the synthesis rate of the different cell components changed depending on position in cell cycle. The growth process may be divided into two phenomena: the formation of new cells during mitosis with a low yield, and size increase of new born cells with high yield. Both kinetic and stoichiometric coefficients varied with the position in the oscillation: the results showed that biomass structure changed and that specific growth rate, as well as biomass yield, varied by +/- 25% during the oscillation.     

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: modeling the elution of DNA from nucleoids

A 36-year-old male with unspecific symptoms and normal physical examination had right cardiac enlargement on chest X-ray. Two-dimensional echocardiographic and thoracic computed tomography demonstrated an intracardiac mass. The tumor was surgically resected and the pathological diagnosis was mixed-type epicardial hemangioma. We discuss this case and review the literature.     

Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.