On the fidelity of DNA polymerase alpha: the influence of alpha-thio dNTPs, Mn2+ and mismatch repair.

The phi X174am16 revertant system has been used to investigate the influence of alpha-thio-dNTPs and of Mn2+ on the fidelity of the 9S DNA polymerase alpha from calf thymus. Upon substituting dGTP by alpha-thio-dGTP during the in vitro replication, a nearly tenfold decrease in the frequency of G:G and G:T mispairs is observed. The formation of all other mispairs is not changed in the presence of the corresponding alpha-thio-dNTP. Mn2+ at concentrations of 0.5 mM does not influence the frequencies of the mispairs. The expression rate of errors formed during in vitro replication in the (-) strand has been determined for all mispairs detectable in the phi Xam16 system. The (-) strand expression of G:T, T:T and C:T mismatches is about 50%, whereas for A:G, G:G and C:A mismatches it is clearly below 50%. We conclude that the different base-base mismatches are repaired with different efficiencies.     

Accuracy of DNA polymerase-alpha in copying natural DNA

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.     

Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.     

Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro

Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool. We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells. By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations. However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation. During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated. Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low [dGTP]. An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency. We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP). This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.     

Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand.

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

Fidelity of DNA replication under conditions used for oligodeoxynucleotide-directed mutagenesis

The fidelity of DNA replication in vitro by DNA polymerase I (large subfragment) of Escherichia coli has been measured by the standard bioassay: single-stranded φX174 DNA (plus strand) containing an amber codon was primed with a synthetic oligodeoxynucleotide, replicated and the frequency of point mutations formed in the synthetic minus strand of the resultant double-stranded DNA determined from the number of revertant phage produced in a spheroplast assay. Since the assay depends crucially on the frequency of expression of the mutations in the heteroduplex, and this can vary for a variety of reasons, parallel control experiments were performed using a primer that covered the amber codon but contained the same mismatch that occurred during replication. The frequency of expression of these mutations was found to vary from 40 to 100% in fully ligated heteroduplexes, depending upon the age and batch of spheroplasts used. The variation probably reflects the viability of the post-replicative mismatch repair enzymes in the spheroplasts used for transfection. Far lower frequencies of expression were found under conditions of poor replication. Accurate data and rate laws for fidelity are obtained only when the bioassay is normalized for the variation in the expression frequency.There is active proofreading by the 3′-5′-exonuclease activity of the polymerase of a misincorporation resulting from a dGTP: T mismatch. The contribution of proofreading to fidelity is low: accuracy is enhanced by a factor of less than 7 at the concentrations of dNTPs in vivo. The lower accuracy of Pol I than Pol III is due mainly to poorer proofreading, which is manifested in a lower “cost” of replication: only 0.7 to 1.7% of the dNTPs are turned over to dNMPs during replication compared with 6 to 13% for Pol III. The error rates measured for Pol I under conditions used for oligodeoxynucleotide-directed mutagenesis are sufficiently low that extraneous errors should not be induced when the concentrations of dNTPs are balanced. However, even higher fidelity will be obtained using the lowest concentrations of dNTPs consistent with efficient replication (~20 μm). Highly unbalanced concentrations as used in pulsed labelling should be avoided.     

Site-specific gap-misrepair mutagenesis by O4-ethylthymine

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

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.     

Oligodeoxynucleotide-directed cleavage and repair of a single-stranded vector: a method of site-specific mutagenesis

A simple and efficient site-specific mutagenesis method is described. First, a single-stranded (ss) circular vector is linearized at the site where the desired mutation will be introduced. To do this, an oligodeoxynucleotide complementary to the target region of the ss vector and containing a restriction enzyme recognition sequence is annealed to the circular ss vector, and the partial double-strand formed is subsequently cleaved with that enzyme. Then, another oligodeoxynucleotide spanning the nick and carrying the mutation is annealed to the linearized ss DNA template and the annealed mixture is used directly to transform Escherichia coli without prior enzymatic DNA synthesis in vitro. The procedure has been applied successfully to constructing insertion, deletion, and point mutations in both M13 phage vectors and plasmid vectors containing the f1 origin of replication.     

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.     

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.     

DNA synthesis with methylated poly(dC-dG) templates. Evidence for a competitive nature to miscoding by O(6)-methylguanine.

The alternating copolymer poly(dC-dG) has been methylated with either dimethyl sulphate or N-methyl-N-nitrosourea and the levels of the various methylation products determined. In addition to the 3-methylcytosine, 3-methylguanine and 7-methylguanine (produced by both agents) reaction with N-methyl-N-nitrosourea also yielded easily detectable amounts of O(6)-methylguanine and phosphotriesters. These methylated polymers were then used as templates in an in vitro assay with Escherichia coli DNA polymerase I measuring the incorporation of complementary (dCMP and dGMP) and noncomplementary (dAMP and dTMP) nucleotides. When the dimethyl sulphate-methylated polymer was used as template there was virtually no detectable incorporation of non-complementary nucleotides indicating that no miscoding could be attributed to the presence of 3-methylcytosine, 3-methylguanine or 7-methylguanine. However, when the N-methyl-N-nitrosourea-methylated polymer was used as template there was a specific incorporation of dTMP but not of dAMP. The amount of dTMP incorporated was always less than the level of O(6)-methylguanine in the template and was found to vary with the relative concentrations of the deoxynucleoside 5'-triphosphates in the assay. As the amount of dCTP present in the assay was decreased the wrong incorporation of dTMP increased and approached the level that would have been expected for a one-to-one miscoding by O(6)-methylguanine as the concentration of dCTP approached zero. The results indicate that O(6)-methylguanine is capable of miscoding with a DNA polymerase but the miscoding is competitive with the normal incorporation of dCMP: when the 5'-triphosphate precursors are present in equal amounts approximately one O(6)-methylguanine in three miscodes leading to the incorporation of dTMP.     

DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase.

Studies in eucaryotic cells (mainly animals and yeast) indicate that at least two DNA polymerases are involved in DNA replication at the level of the replication fork: DNA polymerase alpha, which is associated with DNA primase, is involved in the replication of the lagging strand; DNA polymerase delta, associated with an exonuclease activity, synthesizes the forward continuous DNA strand. Much less information exists concerning plant systems. Previous work from this laboratory provided preliminary evidence of an association between DNA polymerase B from wheat embryo and an exonucleolytic activity. In this paper, we present additional data on the biochemical properties of DNA polymerase B. An improved purification procedure described in this article has been developed. During all the purification steps the nuclease activity was associated with DNA polymerase activity. A biochemical study of this enzyme activity shows that it is an exonuclease which hydrolyses DNA in the 3' to 5' direction. Moreover, this exonuclease confers a proofreading function to DNA polymerase B. Comparison of DNA polymerase B properties (template specificity, sensitivity to DNA replication inhibitors like aphidicolin and butyl-phenyl dGTP, copurification of DNA polymerase and exonuclease activities) with those of animal DNA polymerase delta indicates that these enzymes share many common features. To our knowledge, this is the first report of DNA polymerase delta in higher plants.     

Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase.

To evaluate the relative contributions of DNA polymerase alpha and DNA polymerase delta in chromosome replication during the S phase of the cell cycle, we have used the permeable cell system for replication as a functional assay. We carried out the analysis of DNA polymerases both in quiescent cells stimulated to proliferate and progress through the cell cycle (monolayers) and in actively growing cells separated into progressive stages of the cell cycle by centrifugal elutriation (suspension cultures). DNA polymerase alpha was measured by using the inhibitor butylphenyl dGTP at low concentrations. Using several inhibitors such as aphidicolin, ddTTP and butylphenyl dGTP, we found that DNA polymerase alpha and delta activity were coordinately increased during S phase and declined at the end. However, DNA polymerase delta was performing about 80% of the total replication and DNA polymerase alpha performed only 20%. This high ratio of DNA polymerase delta to DNA polymerase alpha replication activity was maintained throughout S phase in two entirely different experimental approaches.     

Early dissociation of nuclear factor I from the origin during initiation of adenovirus DNA replication studied by origin immobilization.

The DNA-binding domain of Nuclear Factor I (NFIBD) enhances initiation of adenovirus DNA replication up to 50-fold by binding to the auxiliary region of the origin and positioning the viral DNA polymerase. To study if and when NFIBD dissociates from the template, we immobilized origin DNA to glutathione-agarose beads by means of a GST-NFIBD fusion protein. This immobilized template is active in replication. By analyzing the release of prelabeled templates from the beads under different conditions, we show that NFIBD dissociates already early during initiation. During preinitiation NFIBD remains bound, but as soon as dCTP, dATP or dTTP are added, efficient dissociation occurs. A much lower dissociation level was induced by addition of dGTP. Since dCTP, dATP and dTTP are required for formation of a pTP-CAT initiation intermediate, we explain our results by conformational changes occurring in the polymerase during initiation leading to disruption of both the interaction between the polymerase and NFI as well as the interaction between NFI and the DNA.     

Inhibition of adenovirus DNA replication by vesicular stomatitis virus leader RNA.

Vesicular stomatitis virus (VSV) leader RNA and a synthetic oligodeoxynucleotide of the same sequence were found to inhibit the replication of adenovirus DNA in vitro. In contrast, the small RNA transcribed by the VSV defective interfering particle DI-011 did not prevent adenovirus DNA replication. The inhibition produced by leader RNA was at the level of preterminal protein (pTP)-dCMP complex formation, the initiation step of adenovirus DNA replication. Initiation requires the adenovirus pTP-adenovirus DNA polymerase complex (pTP-Adpol), the adenovirus DNA-binding protein, and nuclear factor I. Specific replication in the presence of leader RNA was restored when the concentration of adenovirus-infected or uninfected nuclear extract was increased or by the addition of purified pTP-Adpol or HeLa cell DNA polymerase alpha-primase to inhibited replication reactions. Furthermore, the activities of both purified DNA polymerases could be inhibited by the leader sequence. These results suggest that VSV leader RNA is the viral agent responsible for inhibition of adenovirus and possibly cellular DNA replication during VSV infection.