Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.     

Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli

To test whether the identity of divalent metal activator affects the specificity of misincorporation during polymerization catalyzed by E. coli DNA polymerase I, we carried out the following procedure. A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5'-triphosphates (dNTPs) until one or a few misincorporations occurred in each elongated primer. The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg2+ or Mn2+) were then isolated and sequenced by the 'dideoxy' chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original 'minus' reactions, activated by Mg2+ or Mn2+. The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn2+ for Mg2+ depended on the nucleotide sequence of the template. At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn2+ versus Mg2+. Of these 8 sites, 4 exhibited increased transversions in the presence of Mn2+, while 4 exhibited decreased transversions with Mn2+.     

Mechanisms of error discrimination by Escherichia coli DNA polymerase I

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., & So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3'- to 5'-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 mM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3'- and 5'-exonuclease activities of DNA polymerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

Kinetics of base misinsertion by DNA polymerase I of Escherichia coli

A simple kinetic analysis of the values of kcat and KM for base insertion and misinsertion during DNA replication is presented and applied to the problem of base misinsertion by DNA polymerase I of Escherichia coli. The role of minor tautomeric forms of deoxynucleoside triphosphates (dNTPs) in purine x pyrimidine mismatching has been examined and it has been shown that the misinsertion frequency via this route should be close to the tautomerization constant in solution and is independent of any effect of the polymerase on the tautomerization of a dNTP when bound. Kinetic data on purine x pyrimidine mismatching indicate that the dNTP in a polymerase-DNA-mismatched-dNTP complex is predominantly in the major tautomeric form. The mutagenic effect of Mn2+ in DNA replication is shown to be mediated by decreasing the values of kcat/KM for the insertion of correct dNTPs, whilst the values of this rate constant for misinsertion are relatively unaffected or increased.     

Recognition of B and Z forms of DNA by Escherichia coli DNA polymerase I

Since the substrate binding domain of the large proteolytic fragment of Escherichia coli DNA polymerase I has been shown to interact with the B forms of DNA, we have studied the ability of this enzyme to recognize structures other than the B form. The polymerase activity has been used to evaluate the degree of recognition of the B and Z forms of DNA. The Z form was found to promote less activity, indicating the probable inability of the polymerase to move along the conformationally rigid form of the template. The present study indicates that the Z-DNA found in vivo may have a role in the control of replication.     

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.     

Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli

DNA polymerase I (Klenow fragment) of Escherichia coli catalyzes the addition of deoxynucleotides to 3' hydroxyl termini of blunt-ended DNA fragments. The product of the reaction, which we call +1 addition, is found only in very low yield under conditions that permit editing by the 3'----5' exonuclease activity of the wild-type polymerase. A mutant form of the Klenow fragment that lacks detectable 3'----5' exonuclease activity shows an elevated accumulation of the +1 addition product. The mutant enzyme can use any one of the four dNTPs to carry out the reaction when each precursor is provided individually. However, in the presence of all four dNTPs the addition of dATP is strongly preferred. Suppression of the editing function of the wild-type polymerase through the use of high concentrations of exogenous deoxynucleoside monophosphates also results in a significant increase in the amount of +1 addition product formed. The presence of a high dNMP concentration also alters the specificity of the nucleotide addition reaction carried out by the wild-type enzyme. Thus, in addition to dATP, the dNTP which is complementary to the exogenous deoxynucleoside monophosphate, is also used in the +1 addition reaction. A similar effect of dNMPs on the specificity of nucleotide addition was obtained with the mutant Klenow fragment. These results define two pathways for the +1 addition reaction: one that does not require coding information from the DNA template and a second in which coding information is provided by the exogenous dNMP.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

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 enzyme's 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.     

Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.     

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.     

Trimethoprim-induced DNA polymerase I deficiency in Escherichia coli K-12

Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase. Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain. These colonies therefore had PolA- phenotypes. Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them. Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.     

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.     

DNA polymerase I modulates inducible stable DNA replication in Escherichia coli.

Mutants of Escherichia coli lacking RNase HI activity and cells induced for the SOS response express modes of DNA replication independent of protein synthesis, called constitutive and induced stable DNA replication, respectively. We report here that mutants deleted for the polA gene express induced stable DNA replication at approximately 25-fold the rate of wild-type cells, whereas constitutive stable DNA replication is not enhanced.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.