Piotr Jonczyk

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair‐defective strains containing the polBex1 allele – encoding a polymerase‐proficient but exonucleolytically defective Pol II – displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading‐ versus lagging‐strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE‐produced misinsertion errors. A second role of Pol II is to protect mismatched 3′ termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

The gyrB gene product functions in both initiation and chain polymerization of Escherichia coli chromosome replication: suppression of the initiation deficiency in gyrB-ts mutants by a class of rpoB mutations

SummaryA class of rpoB mutations is described which suppresses replication and transcription deficiency in gyrB-ts mutants shifted to a nonpermissive temperature. The compensatory effect of an altered subunit B of RNA polymerase (rpoB) for the gyrB defect, indicates that transcription is a primary target of the B subunit of DNA gyrase. One gyrB mutation (gyrB402-ts) shows deficiency in chromosome elongation at the nonpermissive temperature, both in vivo and in cells permeabilized with toluene. It is therefore concluded that the gyrB polypeptide functions at least dually in replication; first, at the level of transcription initiation and second, at the level of chain polymerization.

Mutator Phenotype Resulting from DNA Polymerase IV Overproduction in Escherichia coli: Preferential Mutagenesis on the Lagging Strand

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli.

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.

Role of DNA Polymerase IV in Escherichia coli SOS Mutator Activity

Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).

Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae.

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3′ → 5′ proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3′ → 5′ exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli: Analysis of the dnaX36 Mutator Mutant

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (-1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the tau subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of tau, essential for interaction of tau with the alpha (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered alpha-tau interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of tau subunit, in securing a high fidelity of replication.

Role of Escherichia coli DNA Polymerase I in chromosomal DNA replication fidelity

We have investigated the possible role of Escherichia coli DNA polymerase (Pol) I in chromosomal replication fidelity. This was done by substituting the chromosomal polA gene by the polAexo variant containing an inactivated 3′→5′ exonuclease, which serves as a proofreader for this enzymes misinsertion errors. Using this strain, activities of Pol I during DNA replication might be detectable as increases in the bacterial mutation rate. Using a series of defined lacZ reversion alleles in two orientations on the chromosome as markers for mutagenesis, 1.5‐ to 4‐fold increases in mutant frequencies were observed. In general, these increases were largest for lac orientations favouring events during lagging strand DNA replication. Further analysis of these effects in strains affected in other E. coli DNA replication functions indicated that this polAexo mutator effect is best explained by an effect that is additive compared with other error‐producing events at the replication fork. No evidence was found that Pol I participates in the polymerase switching between Pol II, III and IV at the fork. Instead, our data suggest that the additional errors produced by polAexo are created during the maturation of Okazaki fragments in the lagging strand.

Lack of Strand Bias in UV-Induced Mutagenesis in Escherichia coli

We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.

Essential role of the gyrB gene product in the transcriptional event coupled to dnaA-dependent initiation of Escherichia coli chromosome replication

SummaryWhen a culture of the gyrB41-ts mutant is shifted to the nonpermissive temperature, DNA synthesis is arrested at the initiation phase of chromosome replication. After thermal inactivation of the gyrB gene product reinitiation occurs in the presence of chloramphenicol but not in the presence of rifampicin. This suggests that the B subunit of DNA gyrase may regulate synthesis of an “initiator RNA”. An rpoB202 mutation has been isolated which suppresses both the DnaA-initiation phenotype and the inihibitory action of antibiotics which are known to result in relaxation of chromosomal DNA in vivo. We propose that DNA tertiary structure rather than DNA gyrase itself plays an essential regulatory function in the dnaA-dependent transcription which precedes the initiation of chromosome replication.