Yeast DNA Research Articles

Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT(P)) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT(P) loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.

Mathematical Analysis of Genomic Evolution

Changes in nucleotide sequences, or mutations, accumulate from generation to generation in the genomes of all living organisms. The mutations can be advantageous, deleterious, or neutral. The goal of this project is to determine the amount of advantageous mutations it takes to get human (Homo sapiens) DNA from the DNA of genetically distinct organisms. We do this by collecting the genomic data of such organisms, and estimating the amount of mutations it takes to transform yeast (Saccharomyces cerevisiae) DNA to the DNA of a human. We calculate the typical number of mutations occurring annually through the organism's average life span and the average mutation rate. This allows us to determine the total number of mutations as well as the probability of advantageous mutations. Not surprisingly, this probability proves to be fairly small. A more precise estimate can be determined by accounting for the differences in the chromosomal structure and phenomena like horizontal gene transfer.

Mechanism for priming DNA synthesis by yeast DNA Polymerase α

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI:http://dx.doi.org/10.7554/eLife.00482.001.

Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair

The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (K(D) ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nM, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms.

An easy, quick and cheap high-throughput method for yeast DNA extraction from microwell plates

We herein present a high-throughput and cheap method for yeast DNA isolation in a 96 well microplate. About1500 yeast isolates can be processed within one working day and final DNA concentrations are suitable for direct application in PCR-based molecular typing methods.

Идентификация геновSaccharomyces cerevisiae, влияющих на синтетическую летальность приона [PSI+] с мутациями в генеSUP45

Previously, we proposed a test system allowing to perform search for genes that influence the properties of the Sup35 and Sup45 protein. This test is based on the phenomenon of lethality of diploids that combine mutations in SUP45 gene with [PSI+] prion. Lethality of this combination depends on the type of sup45 mutation, and the properties of the prion. [PSI+] variant, which is a strong suppressor ([PSI+]s), showing synthetic lethality with all the nonsense mutations and some missense sup45 mutations in the heterozygote state. The presence of extra copies of a gene under test that affects the phenotypic manifestation of prion [PSI+] or translation termination factors properties, leads to the increase or decrease in diploid lethality. Gene library screening using this test system allowed us to establish the effect of ten fragments of genomic DNA of yeast on synthetic lethality. Deletion analysis of these regions has led to the identification of the HLJ1 and TEF2 genes, as affecting Sup35 protein prionization and/or the efficiency of translation termination.

Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase

Telomeres of the fission yeast,  Schizosaccharomyces pombe , are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.

Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase

Telomeres of the fission yeast, Schizosaccharomyces pombe, are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.

Nuclear Repositioning Precedes Promoter Accessibility and Is Linked to the Switching Frequency of a Plasmodium falciparum Invasion Gene

Variation of surface adhesins, such as the Plasmodium falciparum erythrocyte invasion ligand PfRh4, is critical for virulence and immune evasion in many microbes. While phenotypic switching is linked to transcriptional changes and chromatin function, the determinants of switching frequency remain poorly defined. By expressing a prokaryotic DNA methylase in P.falciparum, we directly assayed accessibility of transcriptionally active and silent chromatin at the PfRh4 locus. Parasites selected for invivo PfRh4 activation show a reversible increase in promoter accessibility and exhibit perinuclear repositioning of the locus from a silent to a conserved activation domain. Forced activation of a proximal gene results in a similar repositioning of the PfRh4 locus, witha concomitant increase in PfRh4 activation ina subpopulation of parasites and promoter accessibility correlating with actively transcribed loci. Thus, nuclear repositioning is associated with increased P.falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

Flow manipulation and cell immobilization for biochemical applications using thermally responsive fluids

A flow redirection and single cell immobilization method in a microfluidic chip is presented. Microheaters generated localized heating and induced poly(N-isopropylacrylamide) phase transition, creating a hydrogel that blocked a channel or immobilized a single cell. The heaters were activated in sets to redirect flow and exchange the fluid in which an immobilized cell was immersed. A yeast cell was immobilized in hydrogel and a 4',6-diamidino-2-phenylindole (DAPI) fluorescent stain was introduced using flow redirection. DAPI diffused through the hydrogel and fluorescently labelled the yeast DNA, demonstrating in situ single cell biochemistry by means of immobilization and fluid exchange.

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich's ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

Structural Basis of Recruitment of DNA Polymerase ζ by Interaction between REV1 and REV7 Proteins

REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.

Evaluation of antibacterial, antioxidant and DNA protective capacity of Chenopodium album’s ethanolic leaf extract

We have investigated the antibacterial effects of Chenopodium album’s ethanolic leaf extract (CAE) on all the Gram (+) and Gram (−) microorganisms and evaluated the protective effects of CAE on both yeast and human mononuclear leukocytes’ genomic DNA upon oxidative shock. Antibacterial activity was recorded on Bacillus subtilis with 13mm of inhibition zone. Total oxidative status (TOS) and the total antioxidative status (TAS) levels were determined to evaluate the antioxidant activity of CAE. Results indicated that there was a good correlation between dose of CAE and TAS levels. We also observed that CAE protect the DNA of both yeast and mononuclear leukocytes against the damaging effect of hydrogen peroxide. The comet assay, applied on both Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and human leukocytes, results suggested that there was statistically significant correlation between CAE dilutions and antigenotoxic activity.

Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry.

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3-8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast.

Accurate and Efficient Bypass of 8,5′-Cyclopurine-2′-Deoxynucleosides by Human and Yeast DNA Polymerase η

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5' nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

Dynamics of plant histone modifications in response to DNA damage

DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.

Pol31 and Pol32 subunits of yeast DNA polymerase δ are also essential subunits of DNA polymerase ζ

Replication through a diverse array of DNA lesions occurs by the sequential action of two translesion synthesis (TLS) DNA polymerases (Pols), in which one inserts the nucleotide opposite the lesion and the other carries out the subsequent extension. By extending from the nucleotide inserted by another Pol, Polζ plays an indispensable role in mediating lesion bypass. Polζ comprises the Rev3 catalytic and Rev7 accessory subunits. Pol32, a subunit of the replicative polymerase Polδ, is also required for Polζ-dependent TLS, but how this Polδ subunit contributes to Polζ function in TLS has remained unknown. Here we show that yeast Polζ is a four-subunit enzyme containing Rev3, Rev7, Pol31, and Pol32; in this complex, association with Pol31/Pol32 is mediated via binding of the Rev3 C terminus to Pol31. The functional requirement of this complex is supported by evidence that mutational inactivation of Rev3's ability to bind Pol31 abrogates Polζ's role in TLS in yeast cells. These findings identify an unexpected role of Pol31 and Pol32 as two essential subunits of Polζ, and clarify why these proteins are required for Polζ-dependent TLS, but not for TLS mediated by Polη in yeast cells. To distinguish the four-subunit complex from the two-subunit Polζ, we designate the four-subunit enzyme "Polζ-d," where "-d" denotes the Pol31/Pol32 subunits of Polδ.

Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ

We have investigated the ability of the 3′ exonuclease activity of Saccharomyces cerevisiae DNA polymerase ɛ (Pol ɛ) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ɛ proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2–4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ɛ proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an ‘incorrect’ sugar is less efficient than is proofreading of an incorrect base, Pol ɛ does proofread newly inserted rNMPs to enhance genome stability.

Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron–sulfur cluster domain

Dna2 nuclease/helicase is a multitasking protein involved in DNA replication and recombinational repair, and it is important for preservation of genomic stability. Yeast Dna2 protein contains a conserved putative Fe–S (iron–sulfur) cluster signature motif spanning the nuclease active site. We show that this motif is indeed an Fe–S cluster domain. Mutation of cysteines involved in metal coordination greatly reduces not just the nuclease activity but also the ATPase activity of Dna2, suggesting that the nuclease and helicase activities are coupled. The affinity for DNA is not significantly reduced, but binding mode in the C to A mutants is altered. Remarkably, a point mutation (P504S), proximal to the Fe–S cluster domain, which renders cells temperature sensitive, closely mimics the global defects of the Fe–S cluster mutation itself. This points to an important role of this conserved proline residue in stabilizing the Fe–S cluster. The C to A mutants are deficient in DNA replication and repair in vivo, and, strikingly, the degree to which they are defective correlates directly with degree of loss of enzymatic activity. Taken together with previous results showing that mutations in the ATP domain affect nuclease function, our results provide a new mechanistic paradigm for coupling between nuclease and helicase modules fused in the same polypeptide.