©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Relating Structure to Function in 29 DNA Polymerase (*)

Luis Blanco Margarita Salas

From the Centro de Biología Molecular ``Severo Ochoa'' (CSIC-UAM) Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain

INTRODUCTION
A ``Sliding-back'' Mechanism to Initiate TP-primed DNA Replication
Structural Mapping of the Enzymatic Activities of 29 DNA Polymerase
Communication between the N-terminal and C-terminal Domains: Coordination between Synthesis and Degradation
Future Prospects
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

Bacteriophage 29 DNA polymerase, the product of the viral gene 2, was originally characterized as a protein involved in the initiation of 29 DNA replication based on both in vivo(1) and in vitro(2, 3, 4) studies. The cloning of gene 2(5) , the overproduction and purification of its product(6) , and the development of an in vitro system for complete 29 DNA replication (7) allowed the characterization of protein p2 as the viral DNA replicase(8) . This monomeric enzyme, with a molecular mass of only about 66 kDa, catalyzes two distinguishable synthetic reactions: 1) DNA polymerization, as any other DNA-dependent DNA polymerase, with insertion discrimination values ranging from 10^4 to 10^6 and with an efficiency of mismatch elongation 10^5-10^6-fold lower than that of a properly paired primer terminus(9) ; 2) terminal protein (TP) (^1)deoxynucleotidylation, which consists of the formation of a covalent linkage (phosphoester) between the hydroxyl group of a specific serine residue (Ser) in 29 TP and 5`-dAMP, requires the presence of divalent metal ions and is strongly stimulated by the presence of the viral DNA replication origins. By means of this reaction, in which the TP is acting as a primer, 29 DNA polymerase catalyzes the initiation step of 29 DNA replication(5, 8) . In addition to the synthetic activities, 29 DNA polymerase has two degradative activities: 1) pyrophosphorolysis, the polymerization reversal, whose physiological significance is still unclear(10) ; 2) 3`-5`-exonuclease, shown to be involved in a proofreading function(11, 12) . This activity, kinetically characterized using ssDNA as substrate and Mg as metal activator(13) , degrades processively DNA substrates longer than six nucleotides, the catalytic constant being 500 s. When the DNA length is reduced below 6-4 nucleotides, the 29 DNA polymerase-ssDNA complex dissociates at a rate of 1 s.

The multiple enzymatic activities of 29 DNA polymerase (summarized in Table 1) allow this enzyme to be the only polymerase involved in the replication of the 29 genome(7, 14) . Moreover, the enzyme has two intrinsic properties: high processivity (>70 kilobases) and strand displacement ability(15) . Based on this enzymatic potential, complete replication of both DNA strands can proceed continuously from each terminal priming event, without the need of synthesis of RNA-primed Okazaki fragments and making unnecessary the participation of accessory proteins and DNA helicases. The efficiency of the protein-primed initiation reaction is in part guaranteed by the previous formation of a heterodimer between TP and DNA polymerase(16) , whereas the nucleotide specificity, as in normal DNA polymerization, is dictated by the DNA template(17) .




A ``Sliding-back'' Mechanism to Initiate TP-primed DNA Replication

It has been shown that 29 DNA polymerase does not start replication at the first base of the genome but employs the second position from the 3`-end of the template for the initial base pairing and formation of the corresponding TP-dAMP complex at each DNA end. The DNA ends (telomeres) are recovered by a specific mechanism, so called ``sliding-back,'' that is based on a 3`-terminal repetition of two T residues. This reiteration permits, prior to DNA elongation, the asymmetric translocation of the initiation product, TP-dAMP, to be paired with the first T residue(18) . The fact that TP-containing genomes, either from virus or linear plasmids(7) , contain some kind of sequence repetitions at their ends supports the hypothesis that the ``sliding-back'' mechanism could be a common feature of protein-primed replication systems(18) . This proposal has been demonstrated in the case of bacteriophages PRD1 from Escherichia coli and Cp1 from Streptococcus pneumoniae and in adenovirus. PRD1 DNA polymerase initiates replication at the fourth nucleotide of the terminal 3`-CCCC repetition (19) and Cp1 DNA polymerase at the third nucleotide of the terminal 3`-TTT repetition, (^2)the end being recovered in both cases by a ``stepwise sliding-back'' mechanism. Adenovirus DNA polymerase initiates replication at the fourth nucleotide of the terminal repetition 3`-GTAGTA, followed by a preelongation step that originates TP-CAT, the end being recovered by a ``jumping-back'' step (20) .


Structural Mapping of the Enzymatic Activities of 29 DNA Polymerase

The C-terminal Domain of 29 DNA Polymerase

DNA Polymerization

Our structure-function studies of 29 DNA polymerase started when we found three regions of significant amino acid similarity, shared with other DNA polymerases from eukaryotic origin. Interestingly, these segments of similarity served to identify putative DNA polymerases encoded by linear plasmids from eukaryotic organisms, being also present in the DNA polymerase from bacteriophage T4(21) . In good agreement with such a novel eukaryotic filiation, 29 DNA polymerase and T4 DNA polymerase were shown to be sensitive to specific inhibitors of eukaryotic DNA polymerase alpha such as aphidicolin, phosphonoacetic acid, butylanilino-dATP, and butylphenyl-dGTP(21, 22) . These three regions, located in the C-terminal portion of each polypeptide (see Fig. 1A), contained the amino acid motifs ``DX(2)SLYP,'' ``KX(3)NSXYG,'' and ``YXDTDS.'' The results obtained by site-directed mutagenesis at these three motifs of 29 DNA polymerase (23, 24, 25, 26) support the proposal that these three segments, corresponding to motifs A, B, and C, form an evolutionary conserved polymerization active site in several groups of nucleic acid-synthesizing enzymes (27) . Afterward, more detailed amino acid sequence comparisons, facilitated by the increasing number of DNA polymerase sequences available, allowed definition of additional conserved regions and motifs belonging to the C-terminal portion of the eukaryotic type superfamily(28, 29) , whose general conservation among other polymerase families is not clear at present. Two of these motifs, ``TX(2)GR'' and ``KXY,'' have been also studied by site-directed mutagenesis in 29 DNA polymerase(30, 31) . The mutational analysis demonstrated that the C-terminal two-thirds of the 29 DNA polymerase polypeptide constitutes the polymerization domain, containing sites for interaction with the metal activator, dNTPs, and DNA (see Fig. 1B). Thus, three aspartate residues, invariant in all members of the eukaryotic type superfamily, were implicated in metal binding and catalysis at the polymerization active site(23, 25) . These 29 DNA polymerase residues, Asp, belonging to motif ``DX(2)SLYP,'' and Asp and Asp, belonging to motif ``YXDTDS,'' are predicted to form a metal binding tripod, analogous to that formed by Pol I residues Asp, Asp, and Glu, by human immunodeficiency virus-reverse transcriptase residues Asp, Asp, and Asp(32) , and by Pol beta residues Asp, Asp, and Asp(33, 34) . In addition, 29 DNA polymerase residue Arg, forming part of the ``TX(2)GR'' motif, was also proposed to play a role in catalysis of the polymerization reaction(30) . Three tyrosine residues, invariant or highly conserved in the eukaryotic type superfamily, were identified as directly or indirectly involved in interaction with dNTPs: Tyr (motif ``DX(2)SLYP''(24, 25) ), Tyr (motif ``KX(3)NSXYG''(24, 26) ), and Tyr (motif ``YXDTDS''(23) ). Several defects such as an increased K(m) for dNTPs, instability of the incorporated dNTPs, altered sensitivity to dNTP analogs, and reduced selection of the correct dNTPs, could be measured either during DNA polymerization or TP-primed initiation reactions. Tyr and Tyr were also involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication(35) . Eight residues, invariant or highly conserved in the C-terminal domain of eukaryotic type superfamily, have been involved in binding template-primer structures (see Fig. 1B): Ser (motif ``DX(2)SLYP''(25) ), Asn, Gly, and Phe (motif ``KX(3)NSXYG'' (26) ), Thr and Arg (motif ``TX(2)GR''(30) ), and Lys and Tyr (motif ``KXY''(31) ).


Figure 1: Structure-function studies of 29 DNA polymerase. A, relative arrangement of the most conserved regions among prokaryotic and eukaryotic DNA polymerases. The amino acid sequence of 29 DNA polymerase (572 amino acids) is represented by a bar, with the N terminus at the left. Gray and filled-in regions indicate the predicted 3`-5`-exonuclease and DNA polymerization domains, respectively. The area in between these two domains (ct) has been involved in the communication or cross-talk among these two domains. Alternative nomenclature for the regions (boxed) that contain the motifs (44) is indicated. A, B, and C correspond to motifs that are generally conserved among different classes of nucleic acid-synthesizing enzymes(27) . B, proposed role for individual residues forming highly conserved N-terminal and C-terminal motifs of 29 DNA polymerase, as defined by site-directed mutagenesis. Motifs are represented in single-letter notation, where x indicates any amino acid. Alternative residues for a particular position are separated by a bar. A summary of the mutational analysis carried out in 29 DNA polymerase is described in the text.



Structural Mapping of Processive Synthesis by 29 DNA Polymerase

A flexible subdomain of Klenow, which closes the ``primer cleft'' once the DNA is bound to it(36) , was proposed to be mainly responsible for the extent of processivity required by Pol I, a repair enzyme. However, the high processivity required for DNA replication is generally achieved by association of the catalytic subunit with accessory proteins that reduce the rate of dissociation of the enzyme from the DNA, relative to translocation and further nucleotide addition. The fact that 29 DNA polymerase is highly processive in the absence of any accessory protein suggests that this enzyme must have specific binding subdomains involved in processivity. By amino acid sequence comparisons, two large insertions flanking the evolutionary conserved motif ``KX(3)NSXYG'' have been identified in 29 DNA polymerase and in other DNA polymerases catalyzing TP-primed replication, a mechanism involving highly processive synthesis of both DNA strands(21, 37) . The structural mapping of the putative 29 DNA polymerase domain(s) involved in processivity will be carried out by site-directed mutagenesis of the most conserved residues corresponding to these two specific insertions.

It has been described that the binding of 29 DNA polymerase to DNA primer-template structures is largely enhanced by the presence of metal ions known to activate DNA polymerization(30) . This behavior suggests that metal-assisted DNA binding could also increase the efficiency of DNA translocation, thus favoring the processivity of 29 DNA polymerase.

TP-primed Initiation

29 DNA polymerase interacts with TP to form a very stable heterodimer as a prerequisite in the initiation of 29 DNA replication(16) . By extrapolation to the three-dimensional structure of the Klenow fragment of E. coli DNA polymerase I (Pol IK) complexed with DNA(36) , the same cleft involved in binding the double-stranded region (primer cleft) of the replicating DNA molecule is proposed to be also the TP-binding site. In agreement with that, mutations at residues Thr and Arg (motif ``TX(2)GR'') of 29 DNA polymerase parallelly decreased the ability to bind both the TP and template-primer DNA molecules (30) (see Fig. 1B). Moreover, recent results indicate that one of the specific insertions, proposed to form a flexible domain that could be involved in processivity of protein-priming DNA polymerases, appears to have a direct role in TP binding. (^3)Based on our site-directed mutagenesis analysis of 29 DNA polymerase, it can be also concluded that protein-primed initiation and DNA polymerization are both catalyzed at a unique active site, involving the same critical residues and amino acid motifs generally conserved in eukaryotic type DNA polymerases.

The N-terminal Domain of 29 DNA Polymerase

3`-5`-Exonuclease

Based on both amino acid sequence similarities and site-directed mutagenesis studies in 29 DNA polymerase, Bernad et al.(38) proposed that the 3`-5`-exonuclease active site of prokaryotic and eukaryotic DNA polymerases is evolutionary conserved, being formed by three N-terminal amino acid segments (ExoI, ExoII, and ExoIII) that invariantly contain the five critical residues identified in Pol IK, involved in metal binding and 3`-5`-exonuclease catalysis (39) (see Fig. 1A). The validity of this proposal has been confirmed in the case of other prokaryotic and eukaryotic enzymes such as T7, T4, and herpes simplex virus DNA polymerases, E. coli Pol II, Bacillus subtilis Pol III, and cellular DNA polymerases , , and from Saccharomyces cerevisiae (see an excellent review of all these mutagenesis studies by Derbyshire et al.(40) ). A steady-state analysis of mutants at each putative 3`-5`-exonuclease active site residue of 29 DNA polymerase (Asp, Glu^14, Asp, Tyr, and Asp) demonstrated their role in catalysis, supporting the idea that the geometry of the Pol I 3`-5`-exonuclease active site and the two-metal ion mechanism proposed for this enzyme (41) can be extrapolated to 29 DNA polymerase and the rest of proofreading DNA polymerases(13) . In addition to the residues involved in metal binding and catalysis at the 3`-5`-exonuclease active site, other residues appear to be structurally and functionally conserved at the exonuclease domain of most prokaryotic and eukaryotic DNA polymerases. Among them, 29 DNA polymerase residues Thr and Asn, located at the ExoI and ExoII motifs, respectively, act as single-stranded DNA ligands, having a critical role in the stabilization of the frayed primer terminus at the 3`-5`-exonuclease active site (42) (see Fig. 1B).

Strand Displacement

Surprisingly, the mutational analysis of the ExoI, ExoII and ExoIII motifs of 29 DNA polymerase showed that the intrinsic capacity to couple strand displacement to DNA polymerization is also located in the N-terminal domain, somehow overlapping with the 3`-5`-exonuclease active site (9, 43) (Fig. 1A). Our model proposed that the enzyme could make an alternative use of the ssDNA binding site, present at the N-terminal domain, either to bind the 3`-5`-exonuclease substrate or to stabilize the interaction between the polymerase molecule and the DNA strand to be displaced. However, the ssDNA ligands Thr and Asn of 29 DNA polymerase seem to be specialized in the stabilization of the editing complex, not having a role in the strand displacement capacity of the enzyme(42) . Therefore, a dual role in 3`-5`-exonuclease and strand displacement appears to be restricted to residues directly acting as metal ligands (see Fig. 1B), such as residues Asp and Glu^14 of the ExoI motif (DXE), Asp of the ExoII motif (NX(F/Y)D), and Asp of the ExoIII motif (YX(3)D), or likely affecting the metal binding network, such as Tyr of the ExoIII motif (YX(3)D). These data suggest that the interaction with the displaced strand, leading to duplex opening, could be assisted by contacts with the divalent metal ions that hold and orient the ssDNA substrate for exonucleolytic proofreading.

Structural Independence of 29 DNA Polymerase Domains

A C-terminal deletion derivative of 29 DNA polymerase, containing the first 188 N-terminal amino acid residues (including the three Exo motifs), was independently expressed in E. coli cells. As expected from our hypothesis of a modular organization of enzymatic activities in 29 DNA polymerase, analogous to that of the Klenow fragment of DNA polymerase I, this N-terminal domain was devoid of any synthetic activity (TP-primed initiation and DNA polymerization) but retained 3`-5`-exonuclease activity(44) . Recently, a N-terminal deletion derivative of 29 DNA polymerase, lacking the first 188 N-terminal amino acid residues (among them the three Exo motifs), has been independently expressed in E. coli cells. This C-terminal domain retained both synthetic activities, TP-primed initiation and DNA polymerization, but it was devoid of 3`-5`-exonuclease activity. (^4)


Communication between the N-terminal and C-terminal Domains: Coordination between Synthesis and Degradation

As described before, the mutational analysis carried out along the 29 DNA polymerase molecule allowed demonstration of the existence of two structurally independent domains containing the synthetic and degradative activities of this enzyme. However, for an effective proofreading of DNA polymerization errors, a mechanism for coordinating DNA polymerization and DNA excision must exist, relying on a structural and functional communication or cross-talk between the N-terminal and C-terminal domains. The basis of this communication, specially important in the case of processive DNA polymerases, involves the intramolecular switching of the primer terminus between the polymerization and 3`-5`-exonuclease active sites.

By site-directed mutagenesis of 29 DNA polymerase, it has been recently demonstrated that the conserved motif ``YXG(G/A),'' located between the 3`-5`-exonuclease and polymerization domains of eukaryotic type DNA polymerases (Fig. 1), is a DNA binding motif that plays a role in the coordination between DNA synthesis and proofreading. (^5)We propose that residues Tyr and Phe of 29 DNA polymerase are primarily involved in the stabilization of template-primer structures at the polymerization active site, playing also a role in the movement (switching) of the primer terminus between the polymerase and exonuclease active sites. This dual role could be achieved if the ``YXG(G/A)'' motif is involved in a conformational change, triggered by the unstabilization produced by insertion of a mismatched nucleotide. In addition to this motif, other amino acid residues of 29 DNA polymerase have been implicated in stabilization of the primer terminus at both polymerization (Thr, Arg, Lys, and Tyr) and exonuclease (Thr and Asn) active sites, playing in this case an indirect role in the dynamics of DNA interaction required to coordinate polymerization and proofreading.


Future Prospects

In addition to the structure-function studies that are extrapolatable to most DNA-dependent DNA polymerases, one of the main goals of our research is the characterization of the structural basis for the intrinsic high processivity of 29 DNA polymerase. To approach this problem, we will search for specific subdomains that could lead to a proliferating cell nuclear antigen-like topological interaction with DNA. As it has been described for both proliferating cell nuclear antigen (45) and the beta subunit of E. coli DNA polymerase III(46) , such a strong interaction would be dissociated only when reaching a DNA end, as should be the case after completing replication of the linear 29 DNA molecule. Of additional interest is understanding how 29 DNA polymerase is able to use both a protein and DNA as primers, and the dynamics of interactions occurring at the transition between TP-primed initiation and the elongation stage of 29 DNA replication. One of the most intriguing questions is whether, after formation of the TP-dAMP initiation complex, TP and 29 DNA polymerase must dissociate either as a consequence of the special translocation step (``sliding-back''), necessary to accommodate the newly created primer terminus in an adequate position to accept the next incoming dNTP, or after the synthesis of a short DNA suitable to be used as primer.

Attempts to obtain 29 DNA polymerase crystals adequate for x-ray diffraction analysis were not successful so far. Other approaches, such as the crystallization of 29 DNA polymerase complexed with TP, will be also explored to elucidate the structural basis for the vast potential of this monomeric enzyme.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. This investigation has been aided by Research Grant 5R01 GM27242-16 from the National Institutes of Health, by Grant PB93-0173 from Dirección General de Investigación Científica y Técnica, by Grant CHRX-CT 93-0248 from the European Economic Community, and by an institutional grant from Fundación Ramón Areces.

(^1)
The abbreviations used are: TP, terminal protein; ssDNA, single-stranded DNA; Pol, polymerase.

(^2)
A. Martín, L. Blanco, P. García, M. Salas, and J. Méndez, submitted for publication.

(^3)
J. Méndez, J. M. Lázaro, L. Blanco, and M. Salas, unpublished results.

(^4)
V. Truniger, J. M. Lázaro, L. Blanco, and M. Salas, unpublished results.

(^5)
V. Truniger, J. M. Lázaro, M. Salas, and L. Blanco, submitted for publication.


ACKNOWLEDGEMENTS

We thank all colleagues at the laboratory that have contributed to the results presented in this review.


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