Impairment of the non-catalytic subunit Dpb2 of DNA Pol ɛ results in increased involvement of Pol δ on the leading strand.

The generally accepted model assumes that leading strand synthesis is performed by Pol ε, while lagging-strand synthesis is catalyzed by Pol δ. Pol ε has been shown to target the leading strand by interacting with the CMG helicase [Cdc45 Mcm2-7 GINS(Psf1-3, Sld5)]. Proper functioning of the CMG-Pol ɛ, the helicase-polymerase complex is essential for its progression and the fidelity of DNA replication. Dpb2p, the essential non-catalytic subunit of Pol ε plays a key role in maintaining the correct architecture of the replisome by acting as a link between Pol ε and the CMG complex. Using a temperature-sensitive dpb2-100 mutant previously isolated in our laboratory, and a genetic system which takes advantage of a distinct mutational signature of the Pol δ-L612M variant which allows detection of the involvement of Pol δ in the replication of particular DNA strands we show that in yeast cells with an impaired Dpb2 subunit, the contribution of Pol δ to the replication of the leading strand is significantly increased.

Mutation spectrum data for Saccharomyces cerevisiae psf1-1 pol2-M644G mutants.

DNA replication in Saccharomyces cerevisiae and other eukaryotes is performed mainly by polymerase epsilon (Pol ε) on the leading strand and polymerase delta (Pol δ) on the lagging strand. Using a mutant form of a DNA polymerase enables tracking its signature in the replicated DNA. Here, we used the pol2-M644G allele encoding the catalytic subunit of Pol ε to analyse its contribution to DNA replication in yeast with the psf1-1 allele of an essential gene encoding a subunit of the GINS complex. GINS is involved in the recruitment of Pol ε, the major leading strand replicase. Thus, its defective functioning can affect the involvement of Pol ε in DNA replication. Together with Cdc45 and Mcm2-7, GINS forms the CMG helicase complex. Our DNA sequencing data enable the observation of changes in the mutational spectra in the URA3 reporter gene cloned in two orientations regarding the nearest ARS. The data presented in this article support the study "Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex" [1].

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex.

DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2-7-GINS(Psf1-3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1-1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1-1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.

Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase-Pol ε Interaction.

The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3.

Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriTpCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells.IMPORTANCE The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed E. coli strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, orf35 and orf36, were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha-, Beta-, and Gammaproteobacteria, as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis The antibacterial procedure based on conjugation with the use of the orf35- and orf36-deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.

Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism.

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction.

Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators.

Plasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, from Citrobacter freundii, is classified in the IVB subfamily and in the MPFI group, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely, orf35 and orf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the orf35 and orf36 deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication in Agrobacterium tumefaciens, Ralstonia eutropha, and Pseudomonas putida, though its conjugation system is capable of introducing plasmids bearing oriTpCTX-M3 into these bacteria, which are representatives of Alpha-, Beta-, and Gammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.IMPORTANCE Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in Enterobacteriaceae We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.

Diverse roles of Dpb2, the non-catalytic subunit of DNA polymerase ε.

Timely progression of living cells through the cell cycle is precisely regulated. This involves a series of phosphorylation events which are regulated by various cyclins, activated in coordination with the cell cycle progression. Phosphorylated proteins govern cell growth, division as well as duplication of the genetic material and transcriptional activation of genes involved in these processes. A subset of these tightly regulated genes, which depend on the MBF transcription factor and are mainly involved in DNA replication and cell division, is transiently activated at the transition from G1 to S phase. A Saccharomyces cerevisiae mutant in the Dpb2 non-catalytic subunit of DNA polymerase ε (Polε) demonstrates abnormalities in transcription of MBF-dependent genes even in normal growth conditions. It is, therefore, tempting to speculate that Dpb2 which, as described previously, participates in the early stages of DNA replication initiation, has an impact on the regulation of replication-related genes expression with possible implications for genomic stability.

Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint.

To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants.

Omega (ParB) binding sites together with the RNA polymerase-recognized sequence are essential for centromeric functions of the Pωregion in the partition system of pSM19035.

Partition systems contribute to stable plasmid inheritance in bacteria through the active separation of DNA molecules to daughter cells, and the centromeric sequence located either upstream or downstream of canonical partition operons plays an important role in this process. A specific DNA-binding protein binds to this sequence and interacts with the motor NTPase protein to form a nucleoprotein complex. The inc18-family plasmid pSM19035 is partitioned by products of δ and ω genes, with δ encoding a Walker-type ATPase and ω encoding a DNA-binding protein. As the two genes are transcribed separately, this system differs from others in its organization; nonetheless, expression of these genes is regulated by Omega, which also regulates the copy number of the plasmid (by controlling copS gene expression). Protein Omega specifically recognizes WATCACW heptad repeats. In this study, we constructed a synthetic δω operon to enable an analysis of the centromeric functions of Omega-binding sites Pδ, Pω and PcopS, discrete from their promoter functions. Our results show that these three regions do not support plasmid stabilization equally. We demonstrate that the Pω site alone can simultaneously drive the expression of partition genes from the synthetic δω operon and act as a unique centromeric sequence to promote the most efficient plasmid partitioning. Moreover, Pω can support the centromeric function in concert with the synthetic δω operon expressed from a heterologous promoter demonstrating that Pω is the main centromeric sequence of the δ-ω partition system. Additionally, the RNA polymerase-recognized sequence in Pω is essential for its centromeric function.

Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria I. Partition systems.

Low copy number plasmids cannot rely on the random segregation during bacterial cell division. To be stably maintained in the population they evolved two types of mechanisms (i) partition systems (PAR) that actively separate replicated plasmid molecules to the daughter cells and (ii) toxin-andidote systems (TA) that act after cell division to kill plasmid-less cells. Our knowledge of partition systems has been based mainly on analysis of plasmids from Gram-negative bacteria. Now, numerous partition systems of plasmids from Gram-positive bacteria have also been characterized and make significant contribution to our understanding of these mechanisms.

Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria II. Post-segregational killing systems.

Active support is needed for low copy-number plasmids to be stably maintained in bacterial cells. The mechanisms that fulfill this role are (i) partition systems (PAR) acting to separate plasmid molecules to daughter cells and (ii) toxin-andidote (TA) (post-segregational killing-PSK) systems which arrest cell growth until the plasmid reaches the correct copy-number or kill the cells that have not inherited the plasmid. Our knowledge of toxin-antidote systems comes mainly from studies on Gram-negative bacteria. However, some addiction systems of Gram-positive bacteria have been characterized in detail or recently identified. Altogether, they bring new interesting data on toxin-antidote functioning in bacteria.

Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes.

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198-284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A' and B motifs abolish both the dimerization of Delta and its interaction with Omega.

Plasmid pAMI2 of Paracoccus aminophilus JCM 7686 carries N,N-dimethylformamide degradation-related genes whose expression is activated by a LuxR family regulator.

N,N-Dimethylformamide (DMF), a toxic solvent used in the chemical industry, is frequently present in industrial wastes. Plasmid pAMI2 (18.6 kb) of Paracoccus aminophilus JCM 7686 carries genetic information which is crucial for methylotrophic growth of this bacterium, using DMF as the sole source of carbon and energy. Besides a conserved backbone related to pAgK84 of Agrobacterium radiobacter K84, pAMI2 carries a three-gene cluster coding for the protein DmfR, which has sequence similarities to members of the LuxR family of transcription regulators, and two subunits (DmfA1 and DmfA2) of N,N-dimethylformamidase, an enzyme of high substrate specificity that catalyzes the first step in the degradation of DMF. Genetic analysis revealed that these genes, which are all placed in the same orientation, constitute an inducible operon whose expression is activated in the presence of DMF by the positive transcription regulator DmfR. This operon was used to construct a strain able to degrade DMF at high concentrations that might be used in the biotreatment of DMF-containing industrial wastewaters. To our knowledge, this is the first study to provide insights into the genetic organization and regulation as well as the dissemination in bacteria of genes involved in the enzymatic breakdown of DMF.

Characterization of a novel partition system encoded by the delta and omega genes from the streptococcal plasmid pSM19035.

High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, delta and omega, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other theta- and sigma-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene omega. Analysis of mutants of the parA-like gene delta showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the omega gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), delta, and omega promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that delta and omega constitute a novel type of plasmid stabilization system.