Genetic Analysis of the Hsm3 Protein Function in Yeast Saccharomyces cerevisiae NuB4 Complex

In the nuclear compartment of yeast, NuB4 core complex consists of three proteins, Hat1, Hat2, and Hif1, and interacts with a number of other factors. In particular, it was shown that NuB4 complex physically interacts with Hsm3p. Early we demonstrated that the gene HSM3 participates in the control of replicative and reparative spontaneous mutagenesis, and that hsm3Δ mutants increase the frequency of mutations induced by different mutagens. It was previously believed that the HSM3 gene controlled only some minor repair processes in the cell, but later it was suggested that it had a chaperone function with its participation in proteasome assembly. In this work, we analyzed the properties of three hsm3Δ, hif1Δ, and hat1Δ mutants. The results obtained showed that the Hsm3 protein may be a functional subunit of NuB4 complex. It has been shown that hsm3- and hif1-dependent UV-induced mutagenesis is completely suppressed by inactivation of the Polη polymerase. We showed a significant role of Polη for hsm3-dependent mutagenesis at non-bipyrimidine sites (NBP sites). The efficiency of expression of RNR (RiboNucleotid Reducase) genes after UV irradiation in hsm3Δ and hif1Δ mutants was several times lower than in wild-type cells. Thus, we have presented evidence that significant increase in the dNTP levels suppress hsm3- and hif1-dependent mutagenesis and Polη is responsible for hsm3- and hif1-dependent mutagenesis.

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28 gene

DNA polymerase ε (pol ε) participates in the leading DNA strand synthesis in eukaryotes. The catalytic subunit of this enzyme, Pol2, is a fusion of two ancestral B-family DNA polymerases. Paradoxically, the catalytically active N-terminal pol is dispensable, and an inactive C-terminal pol is essential for yeast cell viability. Despite extensive studies of strains without the active N-terminal half (mutation pol2-16), it is still unclear how they survive and what is the mechanism of rapid recovery of initially miserably growing cells. The reason for the slow progress is in the difficultly of obtaining strains with the defect. We designed a robust method for constructing mutants with only the C-terminal part of Pol2 using allele pol2rc-ΔN with optimized codon usage. Colonies bearing pol2rc-ΔN appear three times sooner than colonies of pol2-16 but exhibit similar growth defects: sensitivity to hydroxyurea, chromosomal instability, and an elevated level of spontaneous mutagenesis. UV-induced mutagenesis is partially affected; it is lower only at high doses in some reporters. The analysis of the genomes of pol2rc-ΔN isolates revealed the prevalence of nonsynonymous mutations suggesting that the growth recovery was a result of positive selection for better growth fueled by variants produced by the elevated mutation rate. Mutations in the CDC28 gene, the primary regulator of the cell cycle, were repeatedly found in independent clones. Genetic analysis established that cdc28 alleles single-handedly improve the growth of pol2rc-ΔN strains and suppress sensitivity hydroxyurea. The affected amino acids are located on the Cdc28 molecule’s two surfaces that mediate contacts with cyclins or kinase subunits. Our work establishes the significance of the CDC28 gene for the resilience of replication and predicts that changes in mammalian homologs of cyclin-dependent kinases may play a role in remastering replication to compensate for the defects in the leading strand synthesis by the dedicated polymerase.Author SummaryThe catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable while the inactive C-terminal part is required for viability. The corresponding strains show a severe growth defect, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly produced fast-growing clones. We discovered that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs during evolution by positive selection for a better growth fueled by variants produced by elevated mutation rates. Mutations in the cell cycle-dependent kinase gene, CDC28, can single-handedly improve the growth of strains lacking the N-terminal part of Pol2. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may play a role in response to the defects of active leading strand polymerase.

STUDY OF SACCHAROMYCES CEREVISIAE YEAST STRAIN SENSITIVITY WITH DOUBLE MUTANT RAD2∆HSM3∆ TO METHYLMETHANESULPHONATE

The sensitivity of the yeast strain Saccharomyces cerevisiae with the double mutant rad2∆hsm3∆ to genotoxicants was studied using methyl methanesulfonate as an example, as well as its influence on the possibility of spontaneous mutagenesis, and further use of the strain as a test for determining genotoxicants in the environment.

Assessment of the variability of Elymus caninus (Poaceae) and closely related taxa from Russia and Kazakhstan using ISSR markers

A comparative study of taxa that are morphologically close to Elymus caninus, occurring in the territory of Russia and Kazakhstan, was carried out based on the ISSR molecular fingerprints. Data showed that the studied taxa are groups of individuals phylogenetically closely related to E. caninus. The assumption is confirmed that E. viridiglumis, as an Elymus species, has a polyphyletic origin as a part of microevolutionary processes in populations E. caninus s. l., possibly involving E. mutabilis. For the Caucasian endemic E. prokudinii and Kazakhstan endemic E. goloskokovii, the origin as result of introgression or spontaneous mutagenesis, i.e. a manifestation of the natural intraspecific polymorphism of E. caninus, is also assumed.

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.