We have investigated the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. This possibility of differential replication fidelity arises from the distinct modes of replication in the two strands, one strand (the leading strand) being synthesized continuously, the other (the lagging strand) discontinuously in the form of short Okazaki fragments. We have constructed a series of lacZ strains in which the lac operon is inserted into the bacterial chromosome in the two possible orientations with regard to the chromosomal replication origin oriC. Measurement of lac reversion frequencies for the two orientations, under conditions in which mutations ref lect replication errors, revealed distinct differences in mutability between the two orientations. As gene inversion causes a switching of leading and lagging strands, these findings indicate that leading and lagging strand replication have differential fidelity. Analysis of the possible mispairs underlying each specific base pair substitution suggests that the lagging strand replication on the E. coli chromosome may be more accurate than leading strand replication.The question as to how organisms duplicate their DNA with high accuracy is of fundamental interest. Previous studies have revealed the functioning of at least three separate steps, base selection, proofreading, and DNA mismatch repair, which, by their sequential action, are responsible for the low error rate of Ϸ10 Ϫ10 per base replicated (1, 2). The most detailed information about this process is available for the bacterium E. coli based on both enzymological and genetical data. Replication of the E. coli chromosome is performed by DNA polymerase III holoenzyme, an asymmetric dimeric enzyme composed of 18 subunits (10 distinct) that simultaneously replicates the leading and lagging strand of the replication fork (for review, see ref.3). It contains two polymerase core units, one for each strand, each consisting of three tightly associated subunits, ␣, , and . Of these, ␣ is the polymerase (dnaE gene product), (dnaQ gene product) is a 3Ј 3 5Ј exonuclease that performs an editing function, and is a small subunit of unknown function. Additional components of the holoenzyme include the subunit ( 2 ) that dimerizes the two cores, the ␤ subunit (␤ 2 ) that encircles the DNA and tethers each DNA polymerase to the DNA to ensure high processivity, and the five-subunit ␥ complex (␥, ␦, ␦Ј, , and ) that loads the ␤ rings onto the DNA.With regard to the fidelity of polymerase III holoenzyme, as studied both in vivo and in vitro, the main focus has been on the role of the ␣ and subunits. The ␣ (polymerase) subunit plays a critical role through the process of base selection, selecting with great preference correct nucleotides at the nucleotide insertion step. The subunit, in conjunction with the polymerase, is responsible for the subsequent proofreading step, in which by virtue of its 3Ј exonuclease activity incorrectly inserted ...

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10−9 to 10−11 errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back‐up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging‐strand‐specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.

SummaryEscherichia 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 leadingversus 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.

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 temperaturesensitive 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 39 / 59 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 39 / 59 exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

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.

SummaryThe role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon.

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 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 , essential for interaction of with the ␣ (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered ␣-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 damagerelated 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 subunit, in securing a high fidelity of replication.The mechanisms by which cells produce mutations, or try to avoid making them, are of significant research interest. Mutations may occur from replication errors, as DNA replication proceeds with high but not infinite accuracy. While the fidelity of individual DNA polymerases, including their base insertion fidelity and proofreading ability, has been investigated in detail (for reviews, see references 43 and 44), recent emphasis has shifted to the fidelity of the chromosomal replisomes, the multisubunit complexes that perform the simultaneous replication of leading and lagging strands. Specific issues of interest are the contribution of the various replisomal subunits, the mechanisms underlying the differential fidelity of leading and lagging strand replication, and the involvement of the additional DNA polymerases that have been discovered in recent years.In the model system Escherichia coli, chromosomal replication is performed by the 17-subunit protein complex DNA polymerase III (Pol III) holoenzyme (HE) (49,50,56). HE is organized into several functional modules: two Pol III core units (one for each strand), two ␤-clamp processivity factors, and the DnaX complex. Each Pol III core is made up of three subunits (␣, ε, and ), in which ␣ is the DNA polymerase, ε is the proofreading subunit (3Ј35Ј exonuclease), and is a stabilizing factor for the ε subunit (37, 82). Each ␤-clamp is a dimer of identical subunits (␤ 2 ) in the shape ...