Diploid Yeast Cells Research Articles

Increase in radiosensitivity of cells after inhibition of their ability to repair after potentially lethal irradiation damage

This study presents new experimental data demonstrating the recovery of irradiated cells in a nutrition medium that is usually associated with proliferation retardation. The effect of such recovery on the radiosensitivity of diploid yeast cells of different strains after ionizing and UV irradiation was determined by a previously described method. Observed increase of radiosensitivity was shown to be much higher than expected after the inhibition of recovery after potentially lethal damage. The data show that the inhibition of cells ability to recover after potentially lethal damages after incubation of irradiated cells in a nonnutrition medium can in many cases be an indicator of suppression of the total cells’ ability to recover. Presented experimental data indicate that this rule is not universal.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Alpers disease mutations in human DNA polymerase gamma cause catalytic defects in mitochondrial DNA replication by distinct mechanisms.

The human mitochondrial DNA polymerase gamma (Pol-γ) is nuclearly encoded and is responsible for the replication and repair of the mitochondrial genome. Mutations S305R and P1073L in the POLG gene have been reported to be associated with early childhood Alpers syndrome. One patient harboring both mutations as compound heterozygous died at 2 years of age after disease onset at 9 months. Quantitative kinetic analysis on purified enzyme showed that the S305R mutation reduces the DNA binding affinity by 10-fold, and reduces the specificity constant (kcat/Km) for correct nucleotide incorporation by fourfold. It also causes a ∼threefold reduction in the excision rate to remove mismatched nucleotides. Compared to the wild-type Pol-γ, the S305R mutant showed no product formation in a reconstituted rolling circle replisome assay. Interestingly, the P1073L mutant exhibited wild-type activity in single turnover kinetics to quantify changes in kcat/Km, kcat, kexo, or processivity, and showed a twofold decrease in the net polymerization rate in the reconstituted replisome assay, while in yeast, P1073L caused a 60–70% mtDNA reduction in haploid cells. The heterozygous diploid yeast cells carrying S305R and P1073L mutations in trans showed ∼75% reduction of mtDNA content, relative to homozygous diploid cells with two wild-type alleles. Taken together, we show clearly in both the rolling circle and the humanized yeast system that the P1073L mutation caused significant defects in mtDNA replication, and our results suggest a role for P1073 in the functioning of the Pol-γ with the mitochondrial DNA helicase, and provide a rationale for understanding the physiological consequences of the S305R/P1073L compound heterozygote in humans.

Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development.

Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood.

Radiosensitivity, liquid-holding recovery and relative biological effectiveness of densely-ionizing radiation after repeated irradiation of yeast cells

Experimental results described earlier showed significantly larger relative biological effectiveness (RBE) values for wild-type diploid cells in comparison with radiosensitive mutants. This aspect was further studied in this paper. Diploid yeast cells were irradiated with gamma rays from 60Co and alpha particles from 239Pu in the stationary phase of cell growth. Survival curves and the kinetics of the liquid-holding recovery were measured. When the irradiated cells had completely recovered from potentially lethal damage, they were again exposed to radiation and allowed post-irradiation recovery. The procedure was repeated three times. By use of a quantitative approach – describing the process of recovery as a decrease in the effective radiation dose –, the probability of recovery per unit time and the proportion of irreversibly damaged cells were quantitatively estimated. It was shown that the irreversible fraction of cell injury was increased after repeated exposures to gamma rays, from 0.4 after the first irradiation to 0.7 after the third exposure. The effect was more clearly expressed after exposure to densely ionizing radiation, the corresponding values being 0.5 and 1.0. In contrast, the recovery constant did not depend on the number of repeated irradiations and only slightly depended on radiation quality. It is suggested that the process of recovery from potentially lethal radiation damage itself is not impaired after repeated exposures to both low- and high-LET radiations, and the decrease in the ability of the cell to recover from radiation damage is mainly explained by the increase in the proportion of irreversibly damaged cells.

Protein signatures of oxidative stress response in a patient specific cell line model for autism

BackgroundKnown genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress.MethodsProtein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways.ResultsThe protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects’ and the ASD patients’ set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress.ConclusionsRedox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.

Correlations in Chromatin Movement in Diploid Yeast Revealed by Two-Color Three-Dimensional Single-Particle Tracking using the Double-Helix Point Spread Function (DH-PSF) Microscope

It is known that gene regulation in eukaryotes is intimately tied to the three-dimensional (3D) organization of chromatin within the nucleus, and the exact structure and dynamics of this organization is a current topic of intense investigation. In order to examine whether the activation of two alleles in a diploid genome is coordinated we performed fast (10 Hz), 3D, simultaneous tracking of the two alleles of the GAL locus in diploid budding yeast cells. We did so by labeling one copy of the locus with LacO/LacI-GFP and the other with TetO/TetR-mCherry and recording the resulting fluorescence images in parallel using the DH-PSF microscope, which enabled precise sub-diffraction 3D localization in each channel ( = [14,13,27] nm for GFP and [25,29,46] nm for mCherry). The simultaneously recorded tracks were then registered to within sub-diffraction distances. We then examined the statistics of allele velocity cross-correlation and colocalization in both the transcriptionally active (cells grown in galactose) and repressed (grown in dextrose) states. As a control we labeled and tracked a single allele with both colors of fluorescent proteins integrated ∼12-18 kb apart. The dual-allele cases showed significant subpopulations of positive correlations that were larger than the 90th percentile value of the single-allele case (0.11), comprising 51% (26/51) in dextrose and 40% (34/86) in galactose. The presence of positive correlations indicates that gene copies are often closely associated in space and time, which may be consistent, for instance, with intertwined polymers or a mutual association with external protein machinery. Interestingly, we find both correlated and uncorrelated examples which appear peripherally confined, as the GAL genes are known to preferentially associate with the nuclear periphery upon activation.

Genome-wide mutation avalanches induced in diploid yeast cells by a base analog or an APOBEC deaminase.

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.

Characterization of Chromosome Stability in Diploid, Polyploid and Hybrid Yeast Cells

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.

High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

High-Resolution Genome-Wide Analysis of Irradiated (UV and -Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

High-Resolution Genome-Wide Analysis of Irradiated (UV and -Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

Screening of proteins binding to mouse cytomegalovirus M122 protein from mouse brain cDNA library by yeast two-hybrid system

Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction. Key words: Cytomegalovirus; M122 protein; Yeast two-hybrid system; Protein-protein interaction

Evaluation of the cytotoxic and antimutagenic effects of biflorin, an antitumor 1,4 o-naphthoquinone isolated from Capraria biflora L

Biflorin is a natural quinone isolated from Capraria biflora L. Previous studies demonstrated that biflorin inhibits in vitro and in vivo tumor cell growth and presents potent antioxidant activity. In this paper, we report concentration-dependent cytotoxic, genotoxic, antimutagenic, and protective effects of biflorin on Salmonella tiphymurium, yeast Saccharomyces cerevisiae, and V79 mammalian cells, using different approaches. In the Salmonella/microsome assay, biflorin was not mutagenic to TA97a TA98, TA100, and TA102 strains. However, biflorin was able to induce cytotoxicity in haploid S. cerevisiae cells in stationary and exponential phase growth. In diploid yeast cells, biflorin did not induce significant mutagenic and recombinogenic effects at the employed concentration range. In addition, the pre-treatment with biflorin prevented the mutagenic and recombinogenic events induced by hydrogen peroxide (H(2)O(2)) in S. cerevisiae. In V79 mammalian cells, biflorin was cytotoxic at higher concentrations. Moreover, at low concentrations biflorin pre-treatment protected against H(2)O(2)-induced oxidative damage by reducing lipid peroxidation and DNA damage as evaluated by normal and modified comet assay using DNA glycosylases. Our results suggest that biflorin cellular effects are concentration dependent. At lower concentrations, biflorin has significant antioxidant and protective effects against the cytotoxicity, genotoxicity, mutagenicity, and intracellular lipid peroxidation induced by H(2)O(2) in yeast and mammalian cells, which can be attributed to its hydroxyl radical-scavenging property. However, at higher concentrations, biflorin is cytotoxic and genotoxic.

Yeast Sex: Surprisingly High Rates of Outcrossing between Asci

Background Saccharomyces yeasts are an important model system in many areas of biological research. Very little is known about their ecology and evolution in the wild, but interest in this natural history is growing. Extensive work with lab strains in the last century uncovered the Saccharomyces life cycle. When nutrient limited, a diploid yeast cell will form four haploid spores encased in a protective outer layer called the ascus. Confinement within the ascus is thought to enforce mating between products of the same meiotic division, minimizing outcrossing in this stage of the life cycle.Methodology/Principal FindingsUsing a set of S. cerevisiae and S. paradoxus strains isolated from woodlands in North America, we set up trials in which pairs of asci were placed in contact with one another and allowed to germinate. We observed outcrossing in ∼40% of the trials, and multiple outcrossing events in trials with three asci in contact with each other. When entire populations of densely crowded asci germinated, ∼10–25% of the resulting colonies were outcrossed. There were differences between the species with S. cerevisiae having an increased tendency to outcross in mass mating conditions.Conclusions/SignificanceOur results highlight the potential for random mating between spores in natural strains, even in the presence of asci. If this type of mating does occur in nature and it is between close relatives, then a great deal of mating behavior may be undetectable from genome sequences.

Blunt-ended DNA double-strand breaks induced by endonucleases PvuII and EcoRV are poor substrates for repair in Saccharomyces cerevisiae

Most mechanistic studies of repair of DNA double-strand breaks (DSBs) produced by in vivo expression of endonucleases have utilized enzymes that produce cohesive-ended DSBs such as HO, I-SceI and EcoRI. We have developed systems for expression of PvuII and EcoRV, nucleases that produce DSBs containing blunt ends, using a modified GAL1 promoter that has reduced basal activity. Expression of PvuII and EcoRV caused growth inhibition and strong cell killing in both haploid and diploid yeast cells. Surprisingly, there was little difference in sensitivities of wildtype cells and mutants defective in homologous recombination, nonhomologous end-joining (NHEJ), or both pathways. Physical analysis using standard and pulsed field gel electrophoresis demonstrated time-dependent breakage of chromosomal DNA within cells. Although ionizing radiation-induced DSBs were largely repaired within 4 h, no repair of PvuII-induced breaks could be detected in diploid cells, even after arrest in G2/M. Rare survivors of PvuII expression had an increased frequency of chromosome XII deletions, an indication that a fraction of the induced DSBs could be repaired by an error-prone process. These results indicate that, unlike DSBs with complementary single-stranded DNA overhangs, blunt-ended DSBs in yeast chromosomes are poor substrates for repair by either NHEJ or recombination.

Mitotic gene conversion events induced in G1-synchronized yeast cells by gamma rays are similar to spontaneous conversion events

In a previous study, we mapped spontaneous mitotic reciprocal crossovers (RCOs) in a 120-kb interval of chromosome V of Saccharomyces cerevisiae. About three-quarters of the crossovers were associated with gene conversion tracts. About 40% of these conversion tracts had the pattern expected as a consequence of repair of a double-stranded DNA break (DSB) of an unreplicated chromosome. We test this hypothesis by examining the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic conversion events associated with spontaneous RCOs, confirming our previous conclusion that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome.

The Dance of Chromosomes during DNA Repair

DNA repair is an essential process for preserving genome integrity. Among the various forms of DNA damage, double-strand breaks (DSBs) are the most cytotoxic and genotoxic. To repair them, eukaryotic organisms use homologous recombination (HR): it consists of exchanging DNA strands between the broken DNA and an intact homologous DNA and it is choreographed by multi-protein complexes (1). During HR, the search for an intact homologous sequence among the whole genome is the most enigmatic stage (2). How can two homologous needles find each other in the genomic haystack? Is search the result of diffusion and chance encounters, or is there a search apparatus dedicated to bringing the homologous sequences together? To explore the choreography of the DNA and the recombination proteins during homology search, we developed an in vivo 3-colors assay in diploid yeast cells where 2 homologous chromosomes are fluorescently marked at the same locus (with GFP-Lac and RFP-Tet arrays), as well as recombination factors (CFP-tagged proteins). Using deconvolution microscopy, we tracked the movement of the two chromosomes in 3-dimensions in the absence and in the presence of a unique DSB induced near one of the marked chromosome. In the absence of DSB, we found that homologous chromosomes undergo a constrain Brownian motion with a diffusion coefficient of 4.10−4 μm2/s inside a small region of 300 nm. When a DSB is induced, the two homologous DNA become highly dynamic and homologous pairing occurs within one hour. This work is the first attempt to visualize simultaneously the movement of two homologous sequences in vivo into and out of repair centres. 1. Lisby, M., Barlow, J.H., Burgess, R.C. and Rothstein R. Cell: 118, 699-713, 2004. 2. Barzel, A. and Kupiec, M. Nature: 9, 27-37, 2007.

Identification of HCV core protein binding proteins by yeast two-hybrid

To identify HCV core protein binding proteins. The library was amplified, purified, and then were transformed into yeast strain Y187. The reconstructed plasmid pGBKT7-core was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp. The transformed AH109 mated with Y187 containing the library plasmid. The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-alpha-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5alpha. The plasmids in DH5alpha were extracted and sequenced. Eleven proteins, including chymotrypsinogen B1 precursor, carboxypeptidase A1, trypsinogen 2, chymotryptic peptide C, trypsin 1, carboxypeptidase B1, kinesin superfamily proteins 3B, trypsin 2, mitochondria protein gene, elastase 3A and colipase were found to be able to bind to HCV core protein. Proteins related with metabolism of glucose and lipid may bind to HCV core protein.

MCM-GINS and MCM-MCM interactions in vivo visualised by bimolecular fluorescence complementation in fission yeast

BackgroundEach of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM.ResultsTo investigate interactions between components of the CMG complex, we have used bimolecular fluorescence complementation (BiFC) in the fission yeast Schizosaccharomyces pombe for the first time, to analyse protein-protein interactions between GINS and MCM subunits expressed from their native chromosomal loci. We demonstrate interactions between GINS and MCM in the nuclei of exponentially-growing fission yeast cells and on chromatin in binucleate S-phase cells. In addition we present evidence of MCM-MCM interactions in diploid fission yeast cells. As with GINS-MCM interactions, MCM-MCM interactions also occur on chromatin in S-phase cells.ConclusionBimolecular fluorescence complementation can be used in fission yeast to visualise interactions between two of the three components of the CMG complex, offering the prospect that this technique could in the future be used to allow studies on replication protein dynamics in living S. pombe cells.