Wrong Nucleotides Research Articles

Excision of nucleoside analogs in mitochondria by p53 protein

Nucleoside analogs, used against HIV, can be incorporated into a mitochondrial DNA by DNA polymerase gamma. Both the decrease in mitochondrial DNA and increased mutations of mitochondrial DNA may lead to mitochondrial diseases. The tumor suppressor protein p53 exhibits 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. In the present study, we investigated the ability of p53 to excise incorporated nucleoside analogs from DNA in mitochondria. The functional interaction of p53 and DNA polymerase gamma during the incorporation of nucleoside analog was examined in mitochondrial fractions of p53-null H1299 cells, as the source of DNA polymerase gamma. Primer extension reactions were carried out to elucidate the incorporation and removal of nucleoside analogs. The results demonstrate that the excision of incorporated nucleoside analogs in mitochondrial fractions of H1299 cells increased in the presence of purified recombinant p53, or cytoplasmic extracts of large cell carcinoma 2 cells expressing endogenous wild-type p53 (but not specifically predepleted extracts) or cytoplasmic extracts of H1299 cells overexpressing wild-type p53, but not exonuclease-deficient mutant p53-R175H. The amount of nucleoside analogs incorporated into the elongated DNA with mitochondrial fractions of human colon carcinoma 116 (HCT116)(p53+/+) cells was lower than that of HCT116(p53-/-) cells. Furthermore, mitochondrion-localized elevation of p53 in HCT116(p53+/+) cells, following the irradiation-stress stimuli, correlates with the reduction in incorporation of nucleoside analogs and wrong nucleotides. p53 in mitochondria may functionally interact with DNA polymerase gamma, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.

DNA damage alters DNA polymerase to a form that exhibits increased discrimination against modified template bases and mismatched primers

Human DNA polymerase δ (Pol δ4), a key enzyme in chromosomal replication, is a heterotetramer composed of the p125, p50, p68 and p12 subunits. Genotoxic agents such as UV and alkylating chemicals trigger a DNA damage response in which Pol δ4 is converted to a trimer (Pol δ3) by degradation of p12. We show that Pol δ3 has altered enzymatic properties: it is less able to perform translesion synthesis on templates containing base lesions (O6-MeG, 8-oxoG, an abasic site or a thymine-thymine dimer); a greater proofreading activity; an increased exonuclease/polymerase activity ratio; a decreased tendency for the insertion of wrong nucleotides, and for the extension of mismatched primers. Overall, our findings indicate that Pol δ3 exhibits an enhanced ability for the detection of errors in both primers and templates over its parent enzyme. These alterations in Pol δ3 show that p12 plays a major role in Pol δ4 catalytic functions, and provides significant insights into the rationale for the conversion of Pol δ4 to Pol δ3 in the cellular response to DNA damage.

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η

Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G → T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5′ and three bases 3′ to the cross-link. The most prevalent semitargeted mutation was a C → T transition immediately 5′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G → T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5′ to the lesion was increased and 3′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determine if human polymerase η (hpol η) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol η can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol η was significantly different from that by yeast pol η in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol η opposite cross-linked T was 3−5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol η was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol η was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol η may play a role in correct bypass of the cross-links.

Exploring the role of large conformational changes in the fidelity of DNA polymerase β

The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase beta are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

Structure and Mechanism of DNA Polymerase β

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.

Nucleotide-Induced DNA Polymerase Active Site Motions Accommodating a Mutagenic DNA Intermediate

DNA polymerases occasionally insert the wrong nucleotide. For this error to become a mutation, the mispair must be extended. We report a structure of DNA polymerase beta (pol beta) with a DNA mismatch at the boundary of the polymerase active site. The structure of this complex indicates that the templating adenine of the mispair stacks with the primer terminus adenine while the templating (coding) cytosine is flipped out of the DNA helix. Soaking the crystals of the binary complex with dGTP resulted in crystals of a ternary substrate complex. In this case, the templating cytosine is observed within the DNA helix and forms Watson-Crick hydrogen bonds with the incoming dGTP. The adenine at the primer terminus has rotated into a syn-conformation to interact with the opposite adenine in a planar configuration. Yet, the 3'-hydroxyl on the primer terminus is out of position for efficient nucleotide insertion.

Computer simulations of protein functions: searching for the molecular origin of the replication fidelity of DNA polymerases.

The use of computers to simulate the functions of complex biological macromolecules is essential to achieve a microscopic description of biological processes and to model and interpret experimental data. Here we apply theoretical computational approaches to investigate the fidelity of T7 DNA polymerase, divided into discrete steps that include contributions from substrate binding, pK(a) shifts, and rate constants for the PO bond-breaking and bond-making processes. We begin by defining the discrimination between right and wrong nucleotides in terms of the free energy landscape for the dNMP incorporation reaction. We then use the linear response approximation and the empirical valence bond methods to obtain converging results for the contribution of the binding and chemical steps to the overall fidelity. These approaches are successful in reproducing general trends in the observed polymerase incorporation fidelity. The calculations demonstrate the potential for further integration of theoretical and experimental studies to analyze high- and low-fidelity DNA polymerases.

Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS-induced RNA cleavage.

We have addressed the question whether TFS, a protein that stimulates the intrinsic cleavage activity of the archaeal RNA polymerase, is able to improve the fidelity of transcription in Methanococcus. Using non-specific transcription experiments, we could demonstrate that misincorporation of non-templated nucleotides is reduced in the presence of TFS. A more detailed analysis revealed that elongation complexes containing a misincorporated nucleotide were arrested, but could be reactivated by TFS. RNase as well as exonuclease III footprinting experiments demonstrated that this arrest was not combined with extended backtracking. Analysis of paused elongation complexes demonstrated that TFS is able to induce a cleavage resynthesis cycle in such complexes, which resulted in the accumulation of dinucleotides corresponding to the last two nucleotides of the transcript. Further analysis of cleavage products revealed that, even under conditions that strongly promote misincorporation, still 50% of the released dinucleotides were correctly incorporated. Therefore, we assume that pausing of elongation complexes is an important determinant of TFS-induced RNA cleavage from the 3' end. As the incorporation rate of wrong nucleotides is about 700-fold reduced, it is possible that this delay also provides an appropriate time window for cleavage induction in order to maintain transcriptional fidelity by preventing misincorporation.

Evidence for reduced copying fidelity of DNA polymerases α, δ, and ε from Novikoff hepatoma cells

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.

The fidelity of misinsertion and mispair extension throughout DNA synthesis exhibited by mutants of the reverse transcriptase of human immunodeficiency virus type 2 resistant to nucleoside analogs.

The AIDS-causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV-1 and HIV-2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3'-mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug-resistant mutants of HIV-1 RT, with an increased fidelity of DNA synthesis relative to the wild-type RT. In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug-resistant mutants of HIV-2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74,Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV-1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV-2 RT has fidelity similar to that of the wild-type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV-2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug-resistance, misinsertion and mispair extension in HIV-2 RT in contrast to HIV-1 RT, providing evidence for potential biochemical differences between these two related RT.

Appendix: GEL KINETIC ANALYSIS OF POLYMERASE FIDELITY IN THE PRESENCE OF MULTIPLE ENZYME DNA ENCOUNTERS

(1). Oc-casionally, however, polymerase fidelity is too high or processiv-ity is too low to detect misincorporation under standard SCHconditions (defined by Poisson statistics as ,20% of p/t DNAextended). Greater quantities of p/t DNA can be exposed to SCHconditions by using trap DNA, which sequesters a polymeraseafter it dissociates from a p/t. Alternatively, the reaction can berun into MCH conditions. In this case, more p/t DNA is processedand the incorporation signal (gel bands at or beyond the targetsite) may even be enhanced at the expense of the unincorporatedsignal (gel band before the target site), but the interpretation ofthe band intensity ratios must be re-evaluated.This appendix is concerned with extending the SCH kineticanalysis to MCH conditions. Consequences of MCH are verydifferent for polymerases with and without exonuclease activ-ity and for incorporation of right and wrong nucleotides. Po-lymerases that lack exonuclease activity eventually incorpo-rate a nucleotide (right or wrong) opposite a target site on everyavailable p/t DNA. When incorporation is rare, as for the wrongnucleotide, the ratio of gel band intensities increases in propor-tion to the number of hits. When incorporation is frequent, asfor the right nucleotide, the ratio of band intensities increasesexponentially with the number of hits. Polymerases with exo-nuclease activity establish a steady state distribution betweenincorporation and nonincorporation after a second hit. Atsteady state the gel band intensity ratio is always greater thanfor SCH, but only to the extent that the polymerase refuses toexcise the nucleotide incorporated at the target site. For thewrong nucleotide the enhancement is generally less than afactor of two; for the right one it can be an order of magnitude.The conclusion is that MCH conditions can tractably and use-fully enhance the incorporation of wrong nucleotides, but arebest avoided (and generally unnecessary) for measuring incor-poration of a right nucleotide.The presentation follows. The first section reviews the orig-inal kinetic model and SCH analysis. The second section de-rives expressions for interpreting gel band intensity ratioscaused by multiple completed hits. This section has three parts:one on the case without exonuclease activity, one on the casewith exonuclease activity, and one on the effect of suppressingexonuclease activity with increased concentrations of next cor-rect (rescue) nucleotide. To apply the various MCH expressionsin the interpretation of experimental results, it is essential toaccount for the distribution in the number of hits across thepopulation of p/t DNA in a reaction. This is the topic of the thirdsection. The net result is a set of guidelines and correctionfactors for the practical application and interpretation of MCHconditions in the measurement of polymerase fidelity. Thefourth, and final, section summarizes the conclusions and em-phasizes the underlying assumptions.

The fidelity of 3' misinsertion and mispair extension during DNA synthesis exhibited by two drug-resistant mutants of the reverse transcriptase of human immunodeficiency virus type 1 with Leu74-->Val and Glu89-->Gly.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leu184]RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-262]. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wild-type > [Val74]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.

A Novel Kinetic Analysis to Calculate Nucleotide Affinity of Proofreading DNA Polymerases:: APPLICATION TO φ29 DNA POLYMERASE FIDELITY MUTANTS

Amino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3'-->5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by phi 29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleotide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that phi 29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide.

Mutants affecting nucleotide recognition by T7 DNA polymerase.

Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in misincorporation assays. Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.

Replacement of the conserved G.U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity, and fidelity.

There is a phylogenetically conserved G.U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 degrees C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G.U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence motifs essential to 3‘–>5‘ exonuclease activity of Escherichia coli DNA polymerase II.

Many DNA polymerases have conserved sequences required for 3'-->5' exonuclease activity, which contributes to the accuracy of DNA replication by removing misincorporated nucleotides prior to chain elongation. Using amino acid sequence alignments, we predicted the putative active site of the 3'-->5' exonuclease of Escherichia coli DNA polymerase II. Site-directed mutagenesis at D155A, E157A, D155A/E157A, D228A, Y330F, and D334A, which are in the predicted exonuclease active regions, specifically inactivated 3'-->5' exonucleolytic activity but not DNA-polymerizing activity of E. coli DNA polymerase II. Furthermore, all of the mutants were diminished in the in vitro proofreading ability, as judged by their increased insertion and extension of wrong nucleotides. These findings indicate that the 3'-->5' exonuclease region of the E. coli DNA polymerase II is in the amino-terminal part of the protein, as it is in other DNA polymerases, and are consistent with the proposal of an evolutionary conserved 3'-->5' exonuclease active site in most DNA-dependent DNA polymerases of both prokaryotic and eukaryotic origin by Bernad et al. (Bernad, A., Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228).

DNA synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base

The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined. In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters. The methylated polymers have been ution of complementary and non-complementary nucleotides determined. With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed. The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied. The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.