Correct Base Pairs Research Articles

Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis.

Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Pol kappa. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta. Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus. Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined.

Effect of the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin on proofreading by Escherichia coli DNA polymerase I (Klenow fragment) in different sequence contexts.

Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders. The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo. Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+). For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C). Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side. In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base. Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side. The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.

The effect of minor-groove hydrogen-bond acceptors and donors on the stability and replication of four unnatural base pairs.

The stability and replication of DNA containing self-pairs formed between unnatural nucleotides bearing benzofuran, benzothiophene, indole, and benzotriazole nucleobases are reported. These nucleobase analogues are based on a similar scaffold but have different hydrogen-bond donor/acceptor groups that are expected to be oriented in the duplex minor groove. The unnatural base pairs do not appear to induce major structural distortions and are accommodated within the constraints of a B-form duplex. The differences between these unnatural base pairs are manifest only in the polymerase-mediated extension step, not in base-pair stability or synthesis. The benzotriazole self-pair is extended with an efficiency that is only 200-fold less than a correct natural base pair. The data are discussed in terms of available polymerase crystal structures and imply that further modifications may result in unnatural base pairs that can be both efficiently synthesized and extended, resulting in an expanded genetic alphabet.

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases.

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.

Low Fidelity DNA Synthesis by a Y Family DNA Polymerase Due to Misalignment in the Active Site

Sulfolobus solfataricus DNA polymerase IV (Dpo4) is a member of the Y family of DNA polymerases whose crystal structure has recently been solved. As a model for other evolutionarily conserved Y family members that perform translesion DNA synthesis and have low fidelity, we describe here the base substitution and frameshift fidelity of DNA synthesis by Dpo4. Dpo4 generates all 12 base-base mismatches at high rates, 11 of which are similar to those of its human homolog, DNA polymerase kappa. This result is consistent with the Dpo4 structure, implying lower geometric selection for correct base pairs. Surprisingly, Dpo4 generates C.dCMP mismatches at an unusually high average rate and preferentially at cytosine flanked by 5'-template guanine. Dpo4 also has very low frameshift fidelity and frequently generates deletions of even noniterated nucleotides, especially cytosine flanked by a 5'-template guanine. Both unusual features of error specificity suggest that Dpo4 can incorporate dNTP precursors when two template nucleotides are present in the active site binding pocket. These results have implications for mutagenesis resulting from DNA synthesis by Y family polymerases.

Discrimination against purine-pyrimidine mispairs in the polymerase active site of DNA polymerase I: a structural explanation.

We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.

Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase.

To examine the role of the mitochondrial polymerase (Pol gamma) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU), beta-L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma holoenzyme in single turnover kinetic studies to measure K(d) (K(m)) and k(pol) (k(cat)) to estimate the specificity constant (k(cat)/K(m)) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) >> (+)3TC >> (-)3TC > PMPA > azidothymidine (AZT) >> Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC approximately equal to (-)3TC > CBV > AZT > PMPA approximately equal to d4T >> ddA (ddI) >> ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.

Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity.

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.

Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid

Mutagenesis and/or construction of recombinants by exchange of genomic regions between parental molecules constitute powerful tools for the study of viroids. However, a large proportion of such modifications results in molecules, which have lost their infectivity. Such is the case for a recombinant viroid named CECS, obtained by replacing the right half of a citrus exocortis viroid (CEVd) by the same region from chrysanthemum stunt viroid (CSVd). In an effort to recover viable infectious progeny from this recombinant, tomato plants were inoculated with an Agrobacterium strain carrying a dimer of the CECS viroid in positive orientation under the control of the CaMV 35S promoter. About 20% of the plants treated in this way were found to be infected with a replicating viroid, which was further propagated. Sequence analysis of six cloned full-length cDNAs derived from progeny molecules revealed the presence of mutations as compared with the parental CECS sequence. However, only two types of mutations were consistently recovered in all progeny molecules, the addition of a G in a string of four at positions 70–73, a mutation frequently observed in CEVd isolates and mutations leading to the restoration of the correct base pairing in secondary hairpin II. These results show that agro-infection is a suitable technique for the recovery of viable molecules from non-infectious viroid mutants and confirm that the ability to form secondary hairpin II is a prerequisite for viroid infectivity.

Identification of hydrogen bonds between Escherichia coli DNA polymerase I (Klenow fragment) and the minor groove of DNA by amino acid substitution of the polymerase and atomic substitution of the DNA.

DNA polymerases replicate DNA with high fidelity despite the small differences in energy between correct and incorrect base pairs. X-ray crystallographic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fidelity. The current hypothesis is that polymerases check the geometry of the base pairs through hydrogen bonds and steric interactions with the minor groove of the DNA. The mechanisms by which these interactions are related to catalysis and fidelity are not known. In this manuscript, we have studied these interactions using a combination of site-specific mutagenesis of Escherichia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA. Crystal structures have predicted hydrogen bonds from Arg668 to the terminal base on the primer (P1) and Gln849 to its base pair partner (T1). Kinetic studies, however, have implicated the minor groove of the primer terminus but not its base pair partner as being important to catalysis and fidelity. Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A. Hydrogen bonds from the DNA were probed with three oligodeoxynucleotides which have a guanine or 3-deazaguanine (3DG) at P1, T1, or T2. We found that the pre-steady-state parameter k(pol) was decreased with R668A (40-fold) and Q849A (150-fold) or with 3DG at P1 (300-fold) or T2 (25-fold). When R668A was combined with 3DG at P1 the decrease in rate was only 80-fold, consistent with a hydrogen bond between Arg668 and P1. In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold. Similar reactions between R668A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.

Low fidelity DNA synthesis by human DNA polymerase-eta.

A superfamily of DNA polymerases that bypass lesions in DNA has been described. Some family members are described as error-prone because mutations that inactivate the polymerase reduce damage-induced mutagenesis. In contrast, mutations in the skin cancer susceptibility gene XPV, which encodes DNA polymerase (pol)-eta, lead to increased ultraviolet-induced mutagenesis. This, and the fact that pol-eta primarily inserts adenines during efficient bypass of thymine-thymine dimers in vitro, has led to the description of pol-eta as error-free. However, here we show that human pol-eta copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. Pol-eta lacks an intrinsic proofreading exonuclease activity and, depending on the mismatch, makes one base substitution error for every 18 to 380 nucleotides synthesized. This very low fidelity indicates a relaxed requirement for correct base pairing geometry and indicates that the function of pol-eta may be tightly controlled to prevent potentially mutagenic DNA synthesis.

Enzymatic Repair of 5-Formyluracil: II. MISMATCH FORMATION BETWEEN 5-FORMYLURACIL AND GUANINE DURING DNA REPLICATION AND ITS RECOGNITION BY TWO PROTEINS INVOLVED IN BASE EXCISION REPAIR (AlkA) AND MISMATCH REPAIR (MutS)

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.

DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Klenow fragment-DNA interaction required for the incorporation of nucleotides opposite guanine and O6-methylguanine.

A mechanism by which the Klenow fragment of DNA polymerase I monitors the geometry of the base pairs may involve hydrogen bonds between the polymerase and the minor groove of the nascent base pair. The involvement of the 3-position of guanine in the template strand was examined by synthesizing oligodeoxynucleotides containing guanine and 3-deazaguanine and comparing the steady-state kinetics of the incorporation of all four dNTPs. The Vmax/Km decreased a significant amount (170-fold) only when dCTP was the co-substrate suggesting that a hydrogen bond exists only when the correct base pair is being replicated. This approach was also used to examine how the Klenow fragment interacts with the 3-position of the mutagenic base O6-methylguanine (O6mG). The Vmax/Km for the incorporation of dTTP opposite O6-methyl-3-deazaguanine (O6m3DG) was 1700-fold less than opposite O6mG. In contrast, a small 6-fold increase in Vmax/Km occurred for the incorporation of dCTP opposite O6m3DG relative to O6mG. This result suggests that the hydrogen bond between the Klenow fragment and O6mG is more important in the incorporation of dTTP opposite O6mG and may contribute to the mutagenicity of O6mG.

Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism.

DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in DNA produced by the base excision repair pathway of mammalian cells. Crystal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol beta: the binary complex with a gapped DNA substrate (2.4 A resolution), the ternary complex including ddCTP (2.2 A), and the binary product complex containing only nicked DNA (2.6 A). Upon binding ddCTP to the binary gap complex, the thumb subdomain rotates into the closed conformation to contact the otherwise solvent-exposed ddCTP-template base pair. Thumb movement triggers further conformational changes which poise catalytic residue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thumb returns to the open conformation as in the gapped binary DNA complex, facilitating dissociation of the product. These findings suggest that pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignment of catalytic groups for catalysis (via thumb closure), but incorrect base pairing will not. The structures also reveal that pol beta binds both gapped and nicked DNA with a 90 degrees kink occurring precisely at the 5'-phosphodiester linkage of the templating residue. If the DNA were not kinked in this way, contact between the thumb and dNTP-template base pair, presumably important for the checking mechanism, would be impossible, especially when the gap is but a single nucleotide. Such a 90 degrees kink may be a mechanistic feature employed by any polymerase involved in filling gaps to completion.

DNA Polymerase Action on Oligonucleotide Templates from Human Ha-rasProtooncogene Containing N6-Deoxyadenosine Adducts Derived from Trans Addition of (+)- and (-)-anti-Benzo[c]phenanthrene-3,4-dihydrodiol 1,2-epoxides at Codon 61

Abstract In the present work we have used a DNA polymerase assay to investigate the primer extension with T7 DNA polymerase (Sequenase 2.0) and the Klenow fragment of Escherichia coli DNA polymerase I (exo − KF) on chemically synthesized 21mer templates representing partial sequences of the human Ha-ras protooncogene with site-specifically positioned trans-N 6-dA adducts of (-)- (adduct 1) and (+)-anti-benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (adduct 2) at codon 61 (CA∗G; A∗ indicates the adducted position). With Sequenase 2.0 a complete block of primer extension opposite both adduct 1 and 2 was noted using a 10mer primer reaching the (n-1)-position of the adduct. A detailed analysis revealed that Sequenase 2.0 exclusively allowed the misincorporation of dAMP opposite both adducts. In contrast, exo − KF allowed a significant full-length extension of the primer beyond adduct 2, whereas this process occured only to a very minor extent for adduct 1. Correspondingly, a more intensive block of the polymerization reaction opposite adduct 1 was seen. Analysis of the arrest bands revealed that dAMP was preferentially incorporated opposite to adduct 1; also incorporation of dGMP was observed, but to a minor extent. Similarly, dAMP was also incorporated opposite to adduct 2 along with dTMP, the latter indicating a correct base pairing. Additional experiments with exo − KF using different 11mer primers containing each of the four nucleotides opposite the adduct suggest that the primer extension beyond adduct 2 observed with the 10mer primer is an error-free process.

A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.

When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.

Enthalpy-Entropy Compensation in DNA Melting Thermodynamics

We investigate enthalpy-entropy compensation for melting of nearest-neighbor doublets in DNA. Based on data for 10 normal doublets and for doublets containing a mispaired or analog base, the correlation of delta Szero with delta Hzero follows a rectangular hyperbola. Doublet melting temperature relates linearly to delta Hzero by Tm = T(o) + delta Hzero/a, where T(o) = 273 K and a = 80 cal/mol-K. Thus Tm is proportional to delta Hzero + aTo rather than to delta Hzero alone as previously thought by assuming delta Szero to be constant. The term aTo = 21.8 kcal/mol may reflect a constant enthalpy change in solvent accompanying the DNA enthalpy change for doublet melting and is roughly equivalent to breaking four H-bonds between water molecules for each melted doublet. The solvent entropy change (aTo/Tm) declines with increasing Tm, while the DNA entropy change (delta Hzero/Tm) rises, so the combined DNA + solvent entropy change stays constant at 80 cal/K/mol of doublet. If such constancy in DNA + solvent entropy changes also holds for enzyme clefts as "solvent," then free energy differences for competing correct and incorrect base pairs in polymerase clefts may be as large as enthalpy differences and possibly sufficient to account for DNA polymerase accuracy. The hyperbolic relationship between delta Szero and delta Hzero observed in 1 M salt can be used to evaluate delta Hzero and delta Szero from Tm at lower, physiologically relevant, salt concentrations.

Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase.

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.