[Prokaryotic primases - structure and function]. / Prymazy prokariotyczne ­ budowa i funkcje.

Primases are responsible for the synthesis of a short oligo RNA, which serves as primer for DNA polymerase. Primases play an essential role in the initiation of DNA replication at the origins, in the synthesis of Okazaki fragments and in the restart of stalled replication forks. Prokaryotic primases based on their structure and sequence alignments are classified as a family of DnaG proteins. Primases from this family contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either interacts with a helicase or is itself a DNA helicase. In this review, we are presenting the comparison of the representative primases from bacteria and bacteriophages, their mode of action and their involvement in DNA replication at the replication fork.

Fidelity of DNA replication-a matter of proofreading.

DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2-3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood. While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3'-5' exonuclease activity) in the fidelity of DNA replication and to highlight what is known about switching from polymerase to exonuclease active site.

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.

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket.

Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.

Different behaviors in vivo of mutations in the beta hairpin loop of the DNA polymerases of the closely related phages T4 and RB69.

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a beta hairpin loop (hereafter called the beta loop) in their exonuclease domains that plays an important role in active-site switching. The beta loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the beta-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 beta loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their beta loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.

[DNA replication fidelity]. / Mechanizmy wiernosci replikacji DNA.

DNA replication fidelity plays fundamental role in faithful transmission of genetic material during cell division and during transfer of genetic material from parents to progeny. Replicative polymerases are the main guardian responsible for high replication fidelity of genomic DNA. DNA main replicative polymerases are also involved in many DNA repair processes. High fidelity of DNA replication is determined by correct nucleotide selectivity in polymerase active center, and exonucleolytic proofreading that removes mismatches from primer terminus. In this article we will focus on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding. We will also stress the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.

The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity.

In the family-B DNA polymerase of bacteriophage RB69, the conserved aromatic palm-subdomain residues Tyr391 and Tyr619 interact with the last primer-template base-pair. Tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. The main-chain amide of Tyr391 interacts with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. We investigated the roles of these residues in replication fidelity in vivo employing phage T4 rII reversion assays and an rI forward assay. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so we used an in vitro fidelity assay. Tyr391F/M replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on single-stranded DNA and double-stranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3' primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis.

The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination.

Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.

Clusters of mutations from transient hypermutability.

Collections of mutants usually contain more mutants bearing multiple mutations than expected from the mutant frequency and a random distribution of mutations. This excess is seen in a variety of organisms and also after DNA synthesis in vitro. The excess is unlikely to originate in mutator mutants but rather from transient hypermutability resulting from a perturbation of one of the many transactions that maintain genetic fidelity. The multiple mutations are sometimes clustered and sometimes randomly distributed. We model some spectra as populations comprising a majority with a low mutation frequency and a minority with a high mutation frequency. In the case of mutants produced in vitro by a bacteriophage RB69 mutator DNA polymerase, mutants with two mutations are in approximately 10-fold excess and mutants with three mutations are in even greater excess. However, phenotypically undetectable mutations seen only as hitchhikers with detectable mutations are approximately 5-fold more frequent than mutants bearing detectable mutations, indicating that they arose in a subpopulation with a higher mutation frequency. Excess multiple mutations may contribute critically to carcinogenesis and to adaptive mutation, including the adaptations of pathogens as they move from host to host. In the case of the rapidly mutating riboviruses, the viral population appears to be composed of a majority with a mutation frequency substantially lower than the average and a minority with a huge mutational load.

Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy.

Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZalpha mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased T(template)*G(primer) mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.

Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins.

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-). Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.