The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme

DNA polymerase III holoenzyme (holoenzyme) processively and rapidly replicates a primed single-stranded DNA circle to produce a duplex with an interruption in the synthetic strand. The precise nature of this discontinuity in the replicative form (RF II) and the influence of the 5' termini of the DNA and RNA primers were analyzed in this study. Virtually all (90%) of the RF II products primed by DNA were nicked structures sealable by Escherichia coli DNA ligase; in 10% of the products, replication proceeded one nucleotide beyond the 5' DNA terminus displacing (but not removing) the 5' terminal nucleotide. With RNA primers, replication generally went beyond the available single-stranded template. The 5' RNA terminus was displaced by 1-5 nucleotides in 85% of the products; a minority of products was nicked (9%) or had short gaps (6%). Termination of synthesis on a linear DNA template was usually (85%) one base shy of completion. Thus, replication by holoenzyme utilizes all, or nearly all, of the available template and shows no significant 5'----3' exonuclease action as observed in primer removal by the "nick-translation" activity of DNA polymerase I.     

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.     

The beta subunit dissociates readily from the Escherichia coli DNA polymerase III holoenzyme

Purified DNA polymerase III holoenzyme (holoenzyme) was separated by glycerol gradient sedimentation into the beta subunit and the subassembly that lacks it (pol III). In the presence of ATP, beta subunit dimer dissociated from holoenzyme with a KD of 1 nM; in the absence of ATP, the KD was greater than 5 nM. The beta subunit was known to remain tightly associated in the holoenzyme upon formation of an initiation complex with a primed template and during the course of replication. With separation from the template, holoenzyme dissociated into beta and pol III. Cycling to a new template depended on the reformation of holoenzyme. Holoenzyme was in equilibrium with pol III and the beta subunit in crude enzyme fractions as well as in pure preparations.     

The N-terminal region of DNA polymerase delta catalytic subunit is necessary for holoenzyme function

Genetic and biochemical studies have shown that DNA polymerase δ (Polδ) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Polδ, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase δ (ΔN Polδ) from calf thymus. The ΔN Polδ was stimulated as the full-length Polδ by PCNA in a RF-C-independent Polδ assay. However, when tested for holoenzyme function in a RF-C-dependent Polδ assay in the presence of RF-C, ATP and replication protein A (RP-A), the ΔN Polδ behaved differently. First, the ΔN Polδ lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing ΔN Polδ was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Polδ. The present study provides the first evidence that the N-terminal part of the large subunit of Polδ is involved in holoenzyme function.     

The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli

The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated. It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations (Lasken, R. S., and Kornberg, A. (1987) J. Biol. Chem. 262, 1720-1724). It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration. This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency. These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation. The intracellular concentration of beta 2 was estimated to be 250 nM (Kwon-Shin, O., Bodner, J. B., McHenry, C. S., and Bambara, R. A. (1987) J. Biol. Chem. 262, 2121-2130) and is 15-fold higher than the estimated intracellular concentration of DNA polymerase III holoenzyme (15 nM). This high concentration of beta 2 may be responsible for the observation that very little (if any) bypass of pyrimidine photodimers occurred in vivo when the SOS system was not induced. Moreover, it predicts that bypass synthesis under SOS conditions might be associated with an altered form of the beta subunit.     

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the Polg^A449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.     

The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli

The dnaZ protein has been purified to near-homogeneity using an in vitro complementation assay that measures the restoration of activity in a crude enzyme fraction from the dnaZ mutant deficient in the replication of phi X174 DNA. Over 70-fold overproduction of the protein was obtained with a bacteriophage lambda lysogen carrying the dnaZ gene. The purified protein, under reducing and denaturing conditions, has a molecular weight of 52,000 and appears to be a dimer in its native form. The dnaZ protein is judged to be th 52,000-dalton gamma subunit of DNA polymerase III holoenzyme (McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484) for the following reasons: (i) highly purified DNA polymerase III holoenzyme contains a 52,000-dalton polypeptide and has dnaZ-complementing activity; (ii) the 52,000-dalton polypeptide is associated tightly with the DNA polymerase III holoenzyme and can be separated from the DNA polymerase III core only with severe measures; (iii) no other purified replication protein, among 14 tested, contains dnaZ protein activity; and (iv) the abundance of dnaZ protein, estimated at about 10 dimer molecules per Escherichia coli cell, is similar to that of the DNA polymerase III core. Among several circular templates tested in vitro (i.e. single stranded phi X174, G4 and M13 DNAs, and duplex phi X174 DNA), all rely on dnaZ protein for elongation by DNA polymerase III holoenzyme. The protein acts catalytically at a stoichiometry of one dimer per template.     

RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli

Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme. Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA. Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition. Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication. These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.     

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA.

The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA. Errors leading to a loss of alpha-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86% of the errors whereas base deletions amount to 12%. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.