Amino acid templating mechanisms in selection of nucleotides opposite abasic sites by a family a DNA polymerase

Cleavage of the N-glycosidic bond that connects the nucleobase to the backbone in DNA leads to abasic sites, the most frequent lesion under physiological conditions. Several DNA polymerases preferentially incorporate an A opposite this lesion, a phenomenon termed "A-rule." Accordingly, KlenTaq, the large fragment of Thermus aquaticus DNA polymerase I, incorporates a nucleotide opposite an abasic site with efficiencies of A > G > T > C. Here we provide structural insights into constraints of the active site during nucleotide selection opposite an abasic site. It appears that these confines govern the nucleotide selection mainly by interaction of the incoming nucleotide with Tyr-671. Depending on the nucleobase, the nucleotides are differently positioned opposite Tyr-671 resulting in different alignments of the functional groups that are required for bond formation. The distances between the α-phosphate and the 3'-primer terminus increases in the order A < G < T, which follows the order of incorporation efficiency. Additionally, a binary KlenTaq structure bound to DNA containing an abasic site indicates that binding of the nucleotide triggers a remarkable rearrangement of enzyme and DNA template. The ability to resolve the stacking arrangement might be dependent on the intrinsic properties of the respective nucleotide contributing to nucleotide selection. Furthermore, we studied the incorporation of a non-natural nucleotide opposite an abasic site. The nucleotide was often used in studying stacking effects in DNA polymerization. Here, no interaction with Tyr-761 as found for the natural nucleotides is observed, indicating a different reaction path for this non-natural nucleotide.     

Building better polymerases: Engineering the replication of expanded genetic alphabets

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.     

Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low-dGTP conditions either by correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype—sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low-dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs.     

Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I

Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.     

Sequence analysis of three family B DNA polymerases from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis.

Three family B DNA polymerase genes, designated B1, B2, and B3, were cloned from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis, and sequenced. Deduced amino acid sequences of B1 and B3 DNA polymerases have all exonuclease and polymerase motifs which include critical residues for catalytic activities. Furthermore, a YxGG/A motif, which is located between 3'-5' exonuclease and polymerization domains of family B DNA polymerases, was also found in each of the B1 and B3 sequences. These findings suggested that S. ohwakuensis B1 and B3 DNA polymerases have both exonuclease and polymerase activities. However, amino acid sequence of the B2 DNA polymerase of this organism contains several amino acid substitutions in Pol-motifs, and also lacks Exo-motif I and Exo-motif II. These substitutions and lack of certain motifs raise questions about polymerase and exonuclease activities of the corresponding gene product. The B3 sequence of S. ohwakuensis is more closely related to Pyrodictium, Aeropyrum, and Archaeoglobus DNA polymerase B3 sequences than to the Sulfolobus B3 sequences. Phylogenetic analysis showed that crenarchaeal B1 DNA polymerases are closely related to each other, and suggested that crenarchaeal B3, euryarchaeal family B, and eukaryal epsilon DNA polymerases may be orthologs.     

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these ‘antimutagenic’ changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient ‘mutator’ derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.     

Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase zeta

DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.     

The use of nonnatural nucleotides to probe the contributions of shape complementarity and pi-electron surface area during DNA polymerization

It is widely accepted that the dynamic behavior of DNA polymerases during translesion DNA synthesis is dependent upon the nature of the DNA lesion and the incoming dNTP destined to be the complementary partner. We previously demonstrated that 5-nitro-1-indolyl-2'-deoxyribose-5'-triphosphate, a nonnatural nucleobase possessing enhanced base-stacking abilities, can be selectively incorporated opposite an abasic site (Reineks, E. Z., and Berdis, A. J. (2004) Biochemistry 43, 393-404.). While the enhancement in insertion presumably reflected the contributions of the pi-electrons present in the nitro group, other physical parameters such as solvation capabilities, dipole moment, surface area, and shape could also contribute. To evaluate these possibilities, a series of 5-substituted indole triphosphates were synthesized and tested for enzymatic incorporation into normal and damaged DNA by the bacteriophage T4 DNA polymerase. The overall catalytic efficiency for the insertion of the 5-phenyl-indole derivative opposite an abasic site is several orders of magnitude greater than the insertion of either the 5-fluoro- or the 5-amino-indole derivative. The generated structure-activity relationship indicates that pi-electrons play the largest role in modulating the catalytic efficiency for insertion opposite this nontemplating DNA lesion. Despite the large size of 5-phenyl-indole, the catalytic efficiency for its insertion opposite natural nucleobases is equal to or greater than that of the 5-fluoro- or 5-amino-indole derivatives. The higher catalytic efficiency reflects a higher binding affinity of 5-phenyl-1-indolyl-2'-deoxyribose-5'-triphosphate and suggests that the polymerase relies on pi-electron surface area rather than shape complementarity as a driving force for polymerization efficiency.     

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A‐rule. In this study, we present X‐ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non‐instructive lesions by DNA polymerases at least from this sequence family.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Antimutator Mutations in the α Subunit of Escherichia Coli DNA Polymerase III: Identification of the Responsible Mutations and Alignment with Other DNA Polymerases

The dnaE gene of Escherichia coli encodes the DNA polymerase (α subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.     

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.     

Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs.

A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.     

Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases

Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.     

DNA polymerases and human diseases

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.     

Human DNA polymerase beta mutations allowing efficient abasic site bypass

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.