Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.     

Fidelity of the human mitochondrial DNA polymerase

We have quantified the fidelity of polymerization of DNA by human mitochondrial DNA polymerase using synthetic DNA oligonucleotides and recombinant holoenzyme and examining each of the possible 16-base pair combinations. Although the kinetics of incorporation for all correct nucleotides are similar, with an average Kd of 0.8 microM and an average k(pol) of 37 s(-1), the kinetics of misincorporation vary widely. The ground state binding Kd of incorrect bases ranges from a low of 25 microM for a dATP:A mispair to a high of 360 microM for a dCTP:T mispair. Similarly, the rates of incorporation of incorrect bases vary from 0.0031 s(-1) for a dCTP:C mispair to 1.16 s(-1) for a dGTP:T mispair. Due to the variability in the kinetic parameters for misincorporation, the estimates of fidelity range from 1 error in 3563 nucleotides for dGTP:T to 1 error in 2.3 x 10(6) nucleotides for dCTP:C. Interestingly, the discrimination against a dGTP:T mismatch is 16.5 times lower than that of a dTTP:G mismatch due to a tighter Kd for ground state binding and a faster rate of incorporation of the dGTP:T mismatch relative to the dTTP:G mismatch. We calculate an average fidelity of 1 error in 440,000 nucleotides.     

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.     

Base pair hydrogen bonds are essential for proofreading selectivity by the human mitochondrial DNA polymerase

We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3-6 s(-1) fast phase, 0.16-0.3 s(-1) slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04-0.22 s(-1)), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.     

Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA

Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10(2)-10(3)-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10(2)-10(3)-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n - 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.     

Exonuclease proofreading by human mitochondrial DNA polymerase

We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.     

Role of hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase iota

Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.     

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.     

Error rate and specificity of human and murine DNA polymerase eta

We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.     

Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase

We have examined the fidelity of polymerization catalyzed by the human mitochondrial DNA polymerase using wild-type and exonuclease-deficient (E200A mutation) forms of recombinant, reconstituted holoenzyme. Each of the four nucleotides bind and incorporate with similar kinetics; the average dissociation constant for ground state binding is 0.8 microm, and the average rate of polymerization is 37 x s(-1), defining a specificity constant kcat/Km = 4.6 x 10(7) x m(-1) x s(-1). Mismatched nucleotides show weaker ground-state nucleotide binding affinities ranging from 57 to 364 microm and slower rates of polymerization ranging from 0.013 to 1.16 x s(-1). The kinetic parameters yield fidelity estimates of 1 error out of 260,000 nucleotides for a T:T mismatch, 3563 for G:T, and 570,000 for C:T. The accessory subunit increases fidelity 14-fold by facilitating both ground-state binding and the incorporation rate of the correct A:T base pair compared with a T:T mismatch. Correctly base-paired DNA dissociates from the polymerase at a rate of 0.02 x s(-1) promoting processive polymerization. Thus, the mitochondrial DNA polymerase catalyzed incorporation with an average processivity of 1850, defined by the ratio of polymerization rate to the dissociation rate (37/0.02) and with an average fidelity of one error in 280,000 base pairs.     

A new paradigm for DNA polymerase specificity

We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the rate of the chemistry step from any contribution toward enzyme specificity. Discrimination against mismatches is enhanced by the rapid release of mismatched nucleotides from the ternary E.DNA.deoxynucleoside triphosphate complex and by the use of substrate-binding energy to actively misalign catalytic residues to reduce the rate of misincorporation. Our refined model for enzyme selectivity extends traditional thermodynamic formalism by including substrate-induced structural alignment or misalignment of catalytic residues as a third dimension on the free-energy profile and by including the rate of substrate dissociation as a key kinetic parameter.     

Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 microM(-1) s(-1), a value approximately 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 microM(-1) s(-1), a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.     

Functional human mitochondrial DNA polymerase gamma forms a heterotrimer

Mitochondrial DNA polymerase gamma (pol gamma) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol gamma is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol gamma catalytic subunit with both the wild-type accessory factor, pol gammaB, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol gamma seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals.     

The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4

One of the most common DNA lesions arising in cells is an apurinic/apyrimidinic (AP) site resulting from base loss. Although a template strand AP site impedes DNA synthesis, translesion synthesis (TLS) DNA polymerases can bypass an AP site. Because this bypass is expected to be highly mutagenic because of loss of base coding potential, here we quantify the efficiency and the specificity of AP site bypass by two Y family TLS enzymes, Sulfolobus solfataricus DNA polymerase 4 (Dpo4) and human DNA polymerase eta (Pol eta). During a single cycle of processive DNA synthesis, Dpo4 and Pol eta bypass synthetic AP sites with 13-30 and 10-13%, respectively, of the bypass efficiency for undamaged bases in the same sequence contexts. These efficiencies are higher than for the A family, exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. We then determined AP site bypass specificity for complete bypass, requiring insertion or misalignment at the AP site followed by multiple incorporations using the aberrant primer templates. Although Dpo4, Pol eta, and Klenow polymerase have different fidelity when copying undamaged DNA, bypass of AP sites lacking A or G by all three polymerases is nearly 100% mutagenic. The majority (70-80%) of bypass events made by all three polymerases are insertion of dAMP opposite the AP site. Single base deletion errors comprise 10-25% of bypass events, with other base insertions observed at lower rates. Given that mammalian cells contain five polymerases implicated in TLS, and given that a large number of AP sites are generated per mammalian cell per day, even moderately efficient AP site bypass could be a source of substitution and frameshift mutagenesis in vivo.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.     

Effect of the Y955C mutation on mitochondrial DNA polymerase nucleotide incorporation efficiency and fidelity

The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson's disease. In single-turnover kinetic studies, we show that the Y955C mutation results in a decrease in the maximal rate of polymerization and an increase in the K(m) for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM(-1) s(-1), respectively, representing reductions of 30-, 110-, and 1300-fold, respectively, relative to the value for the wild-type enzyme. The fidelity of incorporation was reduced 6-130-fold, largely because of the significant decrease in the specificity constant for correct dATP:T incorporation. For example, k(cat)/K(m) for forming a TTP:T mismatch was decreased 10-fold from 0.0002 to 0.00002 μM(-1) s(-1) by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to that of the wild type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, the level of incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role for Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of incorporation of dATP opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.     

Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors

Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.     

Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.