Gene duplications in evolution of archaeal family B DNA polymerases.

All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria. Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases. We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S. solfataricus P2. The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S. solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains. We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae. It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S. solfataricus B3 ortholog. Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S. solfataricus P2 B3 and S. shibatae B3 paralogs are related to one of the two DNA polymerases of P. occultum. These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group. Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events.     

Structural basis for uracil recognition by archaeal family B DNA polymerases

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C-->A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.     

A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2'-deoxyguanosine

The harmfulness of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodG) damage resides on its dual coding potential, as it can pair with the correct dCMP (dC) or the incorrect dAMP (dA). Here, we investigate the translesional synthesis ability of family B ϕ29 DNA polymerase on 8oxodG-containing templates. We show that this polymerase preferentially inserts dC opposite 8oxodG, its 3′–5′ exonuclease activity acting indistinctly on both dA or dC primer terminus. In addition, ϕ29 DNA polymerase shows a favoured extension of the 8oxodG/dA pair, but with an efficiency much lower than that of the canonical dG/dC pair. Additionally, we have analysed the role of the invariant tyrosine from motif B of family B DNA polymerases in translesional synthesis past 8oxodG, replacing the corresponding ϕ29 DNA polymerase Tyr390 by Phe or Ser. The lack of the aromatic portion in mutant Y390S led to a lost of discrimination against dA insertion opposite 8oxodG. On the contrary, the absence of the hydroxyl group in the Y390F mutant precluded the favoured extension of 8oxodG:dA base pair with respect to 8oxodG:dC. Based on the results obtained, we propose that this Tyr residue contributes to dictate nucleotide insertion and extension preferences during translesion synthesis past 8oxodG by family B replicases.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases

Although DNA polymerase eta (Pol eta) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Pol eta of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.     

B family DNA polymerases asymmetrically recognize pyrimidines and purines

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.     

Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.     

An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3′–5′ proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA.     

Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase

The PRD1 DNA polymerase is a small multifunctional enzyme containing three major conserved amino acid sequences shared by family B DNA polymerases. Thus, the PRD1 DNA polymerase provides an useful model system with which to study structure-function relationships of DNA polymerase molecules. In order to investigate the functional and structural roles of the highly conserved amino acid sequences, we have introduced mutations into each of the 3 conserved regions of the PRD1 DNA polymerase. Genetic complementation study as well as DNA polymerase assay indicated that each mutation inactivated DNA polymerase catalytic activity, but not the 3' to 5' exonuclease activity.     

A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide

Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of 29 DNA polymerase. The affinity for nucleotides of 29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of 29 DNA polymerase. The results suggest that Lys371 of 29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that 29 DNA polymerase can achieve the closed conformation in the absence of a DNA template.     

A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

   

Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases.     

Family A and family B DNA polymerases are structurally related: evolutionary implications.

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.     

Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography

Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer-template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.     

A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases.

The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes. They were designed on the basis of partial enzyme amino acid sequences. The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa. By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions. By computer-assisted homology search, several sequence similarities among S. solfataricus and family B DNA polymerases were found. In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified. This result suggests that the proofreading domains of all these enzymes are evolutionarily related.     

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.     

Eukaryotic DNA polymerases, a growing family

In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.     

A model for the dynamics of mammalian family X DNA polymerases

Based on available structural studies, a model is presented for polymerization dynamics of mammalian family X DNA polymerases, including polymerases β, λ, μ, and terminal deoxynucleotidyl transferase (TdT). Using the model, distinct polymerization activities and processivities of the four polymerases acting on different forms of DNA substrate are analyzed and studied theoretically. A “gradient” of template dependence of polymerases β, λ, μ, and TdT is well explained. The much higher occurrence frequencies of the −1 frameshift DNA synthesis by pols λ and μ than that by pol β are well explained. The theoretical results on the polymerization processivities are also in agreement with the available experimental data.     

A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.     

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.