Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

New phenotypic characteristics of three tmm alleles in Arabidopsis thaliana

Key message

Three new tmm mutants were isolated and showed differential phenotypes from tmm - 1 , and TMM overexpression led to abnormal leaf trichomes.

Abstract

TOO MANY MOUTH (TMM) plays a significant role in the stomatal signal transduction pathway, which involves in the regulation of stomatal distribution and patterning. Three mutants with clustered stomata were isolated and identified as new alleles of tmm. tmm-4 mutation included a base transversion from adenine to thymidine in position 1,033 of the TMM coding region and resulted in premature termination of translation at position 345 of TMM. tmm-5 had a base transition from cytosine to thymidine in 244 of TMM and translated 82 amino acids before premature termination. tmm-6 mutation took a base transition from guanine to adenine in 463 of TMM and changed a glycine (Gly) to an arginine (Arg) in position 155 of the protein. tmm-6 had an evident reduction of stomatal clusters and fewer stomata in cluster compared with other tmm alleles, possibly due to decreased level of entry divisions in cells next to two stomata or their precursors. tmm-5 and tmm-6 were hypersensitive to abscisic acid (ABA) in seedling growth and seed germination, while tmm-4 was defective in response to ABA during seed dormancy, suggesting that TMM was involved in ABA signaling transduction. Interestingly, overexpression of TMM resulted in the reduction of leaf trichomes and their branches, and this might reveal a new function of TMM in trichome development.     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex

DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ’s biochemical properties including catalytic efficiency, processivity or proofreading activity – it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^? pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono-γ-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Overexpression of A613T and G462T variants of DNA polymerase β weakens chemotherapy sensitivity in esophageal cancer cell lines

Background

Human DNA polymerase β (polβ) is a small monomeric protein that is essential for short-patch base excision repair. It plays an important role in regulating the sensitivity of tumor cells to chemotherapy.

Methods

We evaluated the mutation of polβ in a larger cohort of esophageal cancer (EC) patients by RT-PCR and sequencing analysis. The function of the mutation was evaluated by CCK-8, in vivo tumor growth, and flow cytometry assays.

Results

There are 229 patients with the polβ mutation, 18 patients with A613T mutation, 12 patients with G462T mutation among 538 ECs. Analysis results of survival time showed that EC patients with A613T, G462T mutation had a shorter survival than the others (P < 0.05). CCK-8 and flow cytometry assays results showed the A613T, G462T EC9706 cells were less sensitive than WT cells to 5-FU and cisplatin (P < 0.05). Experiments results in vivo showed that the tumor sizes of A613T and G462T group were larger than WT and polβ^?/? groups (P < 0.05).

Conclusions

In this study, we discovered A to T point mutation at nucleotide 613 (A613T) and G to T point mutation at nucleotide 462 (G462T) in the polβ gene through 538 EC patients cohort study. A613T and G462T variant of DNA polymerase β weaken chemotherapy sensitivity of esophageal cancer.

     

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

Background

The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1 ^-/- mice using a Cre/loxP system.

Results

Both male and female Ehd1 ^-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1 ^-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1 ^-/- adult males were infertile and displayed decreased testis size, whereas Ehd1 ^-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1 ^-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1 ^-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1 ^-/- mice.

Conclusions

Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.     

Effect of proofreading and dam-instructed mismatch repair systems on N4-hydroxycytidine-induced mutagenesis

Summary The role of the proofreading (35 exonuclease) function of T4 DNA polymerase and the mismatch repair system ofE. coli on N⁴-hydroxycytidine (oh⁴Cyd)¹ induced mutagenesis was investigated. oh⁴Cyd-induced mutation is strongly suppressed when the proofreading activity increases as a result of the presence oftsCB87-antimutator polymerase or elevated temperature (43° C vs 30° C). Mutagenic activity of oh⁴Cyd, however, is little, if at all, affected by the presence of thetsLB56 mutator allele of T4 DNA polymerase with suppressed proofreading activity. This leads to the conclusion that oh⁴C nucleotides are not frequently removed by proofreading activity of wild-type T4 DNA polymerase. The number of mutations induced by oh⁴Cyd increases 3- to 5-fold due to damage of the genesmutS,mutL,uvrE, but notmutR.Dam ^- cells are more sensitive to, and hypermutable by, oh⁴Cyd in comparison withdam ⁺ cells. This is compatible with the notion that oh⁴C residues are recognised and excised by mismatch repair enzymes. The results indicate thath neither the proofreading function of T4 DNA polymerase, nor the mismatch repair enzymes, are responsible for the high specificity of oh⁴Cyd which causes ATGC transition.     

The effect of transpiration on selenium uptake and mobility in durum wheat and spring canola

Aims

The objective of this study was to determine the relative importance of transpirational pull, Se speciation, sulfate and species on Se accumulation by plants, in order to determine which of these factors must be considered in the future development of models to predict Se accumulation by plants.

Methods

Seedlings of durum wheat (Triticum turgidum L. var durum cv ‘Kyle’) and spring canola (Brassica napus L. var Hyola 401) were grown hydroponically and exposed to SeO ₄ ^2- (selenate) with or without SO ₄ ^2- (sulfate), or to HSeO ₃ ^- (biselenite) under different transpiration regimes altered through ‘low’ (~50%) or ‘high’ (~78%) relative humidity (RH). Plants were harvested after 0, 8, 16, or 24?h exposures, digested, and analyzed for Se by GFAAS.

Results

Accumulation and distribution of Se by plants is dependent on plant species, Se speciation in the nutrient solution, SO ₄ ^2- competition, and transpiration regimes. Canola accumulated and translocated more Se than wheat. In wheat and canola, the greatest accumulation and translocation of Se occurred when plants were exposed to SeO ₄ ^2- without SO ₄ ^2- compared to solutions of SeO ₄ ^2- with SO ₄ ^2- or HSeO ₃ ^2- . Wheat plants exposed to SeO ₄ ^2- and SO ₄ ^2- had an increased Se accumulation and translocation under increased transpiration rates than when exposed to SeO ₄ ^2- without SO ₄ ^2- or HSeO ₃ ^2- . On the other hand, increases in transpiration increased the translocation of Se to canola shoots when exposed to HSeO ₃ ^- more than any other treatments.

Conclusions

Overall, our results suggest that plant species is the most important factor influencing Se accumulation and translocation, but that these endpoints can be modified by climate and specific soil Se or S content. Models to predict accumulation of Se by plants must consider all of these factors to accurately calculate the mechanisms of uptake and translocation.     

Fine mapping of Co-x,an anthracnose resistance gene to a highly virulent strain of Colletotrichum lindemuthianum in common bean

Key message

The Co - x anthracnose R gene of common bean was fine-mapped into a 58 kb region at one end of chromosome 1, where no canonical NB-LRR-encoding genes are present in G19833 genome sequence.

Abstract

Anthracnose, caused by the phytopathogenic fungus Colletotrichum lindemuthianum, is one of the most damaging diseases of common bean, Phaseolus vulgaris. Various resistance (R) genes, named Co-, conferring race-specific resistance to different strains of C. lindemuthianum have been identified. The Andean cultivar JaloEEP558 was reported to carry Co-x on chromosome 1, conferring resistance to the highly virulent strain 100. To fine map Co-x, 181 recombinant inbred lines derived from the cross between JaloEEP558 and BAT93 were genotyped with polymerase chain reaction (PCR)-based markers developed using the genome sequence of the Andean genotype G19833. Analysis of RILs carrying key recombination events positioned Co-x at one end of chromosome 1 to a 58 kb region of the G19833 genome sequence. Annotation of this target region revealed eight genes: three phosphoinositide-specific phospholipases C (PI-PLC), one zinc finger protein and four kinases, suggesting that Co-x is not a classical nucleotide-binding leucine-rich encoding gene. In addition, we identified and characterized the seven members of common bean PI-PLC gene family distributed into two clusters located at the ends of chromosomes 1 and 8. Co-x is not a member of Co-1 allelic series since these two genes are separated by at least 190 kb. Comparative analysis between soybean and common bean revealed that the Co-x syntenic region, located at one end of Glycine max chromosome 18, carries Rhg1, a major QTL contributing to soybean cyst nematode resistance. The PCR-based markers generated in this study should be useful in marker-assisted selection for pyramiding Co-x with other R genes.     

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis,but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.     

Metabolic flux balance analysis and the in silicoanalysis of Escherichia coliK-12 gene deletions

Background

Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silicorepresentations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA). FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information.

Results

Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coliusing FBA and examined the optimal utilization of the E. colimetabolic pathways as a function of environmental variables. We have used an in silicoanalysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system) essential for aerobic growth of E. colion glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi ^-, zwf, and pta ^-mutant strains were examined in more detail by mapping the capabilities of these in silicoisogenic strains.

Conclusions

We found that computational models of E. colimetabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silicaresults lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: 10.1186/1471-2105-1-1     

Five new galactolipids from the freshwater microalga Porphyridium aerugineum and their nitric oxide inhibitory activity

Chemical investigation of the freshwater rhodophyte microalga Porphyridium aerugineum led to the isolation of five new galactolipids, namely, (2S)-1-O-eicosapentaenoyl-2-O-arachidonoyl-3-O-β-d-galactopyranosylglycerol (1), (2S)-1-O-eicosapentaenoyl-2-O-linoleoyl-3-O-β-d-galactopyranosylglycerol (2), (2S)-1-O-arachidoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (6), (2S)-1-O-eicosapentaenoyl-2-O-arachidoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (7), and (2S)-1-O-eicosapentaenoyl-2-O-linoleoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (8) together with five known galactolipids. The stereo-structures of all new galactolipids were elucidated by spectroscopic analyses and both enzymatic and chemical degradation methods. This is the first report of galactolipids from P. aerugineum. The newly isolated galactolipids showed strong and dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 macrophage cells. Both galactolipids 1 and 2 possessed stronger NO inhibitory activity than N ^G-methyl-l-arginine acetate salt, a well-known NO inhibitor used as a positive control. Further study suggested that these galactolipids inhibit NO production through downregulation of inducible nitric oxide synthase expression.     

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Background

The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved.

Results

Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Δxyl1 background. This resulted in significant increase in the K_M for NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_M for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Δxyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1 as compared to 4.2 mg × (L × h)^-1 for the wild type strain.

Conclusion

Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.     

Induction of only one SOS operon,umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^?) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.