Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m109.007641
ISSN1083-351X
AutoresKausiki Datta, Neil P. Johnson, Vince J. LiCata, Peter H. von Hippel,
Tópico(s)CRISPR and Genetic Engineering
ResumoIn addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis. In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis. Escherichia coli DNA polymerase (DNAP) 2The abbreviations used are: DNAPDNA polymerasepolpolymeraseexoexonucleaseP/Tprimer-templatentnucleotide(s)ssDNAsingle-stranded DNAdsDNAdouble-stranded DNA2-AP2-aminopurineCDcircular dichroism. 2The abbreviations used are: DNAPDNA polymerasepolpolymeraseexoexonucleaseP/Tprimer-templatentnucleotide(s)ssDNAsingle-stranded DNAdsDNAdouble-stranded DNA2-AP2-aminopurineCDcircular dichroism. I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5′ → 3′ exonuclease activity, an intermediate domain that carries the 3′ → 5′ proofreading activity, and a C-terminal domain that is associated with the 5′ → 3′ template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1De Lucia P. Cairns J. Nature. 1969; 224: 1164-1166Crossref PubMed Scopus (430) Google Scholar). An N-terminal deletion mutant of DNAP I, known as the “large fragment” or Klenow form of the enzyme, contains only the polymerase (pol) and the 3′ → 5′ exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3′ → 5′ exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3′-end of the primer (2Brutlag D. Kornberg A. J. Biol. Chem. 1972; 247: 241-248Abstract Full Text PDF PubMed Google Scholar), thus contributing significantly to the overall fidelity of DNA replication (3Kunkel T.A. Cell. 1988; 53: 837-840Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar). As a consequence, the ability of polymerases to “shuttle” the 3′-end of the primer strand efficiently between the pol and the exo sites in order to rectify misincorporation events during polymerization is critical to maintaining the overall accuracy of template-directed replication. Elucidation of the mechanisms of this shuttling and determination of the factors that control the rates (and equilibria) of the active site switching reaction will certainly increase our understanding of fidelity control by DNA polymerases.An early crystallographic study of the Klenow polymerase complexed with fully paired primer-template (P/T) DNA revealed that 3–4 nt of the 3′-primer terminus had been unwound from the template stand and partitioned into the exo site and that an extended single-stranded DNA (ssDNA) binding pocket of the exo site appeared to make position-specific hydrophobic contacts with the unstacked bases at the 3′-end of the primer (4Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar). A separate crystallographic study of an editing complex confirmed that an ssDNA fragment 4 nt in length was bound at the exo site in the same conformation as seen for the single-stranded 3′-primer sequence unwound from P/T DNA (5Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar). A structure of Klenow polymerase with the DNA bound at the pol site has not yet been reported, although such structures have been obtained for other homologous polymerases, including Klentaq (the “large fragment” of Thermus aquaticus (Taq) DNAP), Bacillus stearothermophilus (Bst) “large fragment” polymerase, and the T7 DNAP (6Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 7Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 8Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar), all of which are members of the polymerase family that includes Klenow.The amino acid residues involved in the binding of DNA at the pol site in these polymerases (determined from co-crystal structures) and those of Klenow (determined by site-directed mutagenesis studies (9Polesky A.H. Steitz T.A. Grindley N.D. Joyce C.M. J. Biol. Chem. 1990; 265: 14579-14591Abstract Full Text PDF PubMed Google Scholar, 10Polesky A.H. Dahlberg M.E. Benkovic S.J. Grindley N.D. Joyce C.M. J. Biol. Chem. 1992; 267: 8417-8428Abstract Full Text PDF PubMed Google Scholar)) are highly conserved, suggesting that a similar DNA binding mode at the pol site may apply to all of the DNAP I polymerases. The crystal structure of Klenow revealed that the polymerization domain has a shape reminiscent of a right hand in which the palm, fingers, and thumb domains form the DNA-binding crevice. Structural studies with various DNAP I polymerases in the presence of P/T DNA constructs yielded an “open” binary complex, whereas the addition of the next correct dNTP (as a chain-terminating dideoxy-NTP) resulted in the formation of a catalytically competent “closed” ternary complex (6Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 7Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 8Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar). In the latter complex, the 3′-primer terminus was base-paired with the template DNA, and the templating base was poised for incorporation of the next correct nucleotide. These structures showed that the conformation of the DNA primer terminus bound at the pol site is markedly different from that of the “frayed open” primer observed at the exo site in Klenow (4Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar, 5Freemont P.S. Friedman J.M. Beese L.S. Sanderson M.R. Steitz T.A. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 8924-8928Crossref PubMed Scopus (324) Google Scholar).Although crystallographic studies have provided a wealth of information about the conformations of the DNA substrates bound at the active sites of DNAP, replication itself is a dynamic process (reviewed in Ref. 11Joyce C.M. Benkovic S.J. Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar), and it is critical to be able to distinguish between various forms of DNA-polymerase complexes in solution in order to fully understand the mechanistic details of the replication process. A solution approach used by Millar and co-workers (reviewed in Ref. 12Bailey M.F. Thompson E.H. Millar D.P. Methods. 2001; 25: 62-77Crossref PubMed Scopus (27) Google Scholar) for studying the conformation of DNA in these complexes involved measuring the time-resolved fluorescence anisotropy properties of a dansyl fluorophore attached to a DNA base located 8 bp upstream of the P/T DNA junction. The changes in the lifetime of the fluorophore, which appeared to depend mostly on the local environment occupied by the probe within the protein (i.e. buried versus partially exposed), were correlated with specific binding conformations of the primer to provide an estimate of the fractional occupancy of the pol and the exo sites. Reha-Krantz and co-workers (13Tleugabulova D. Reha-Krantz L.J. Biochemistry. 2007; 46: 6559-6569Crossref PubMed Scopus (22) Google Scholar) more recently used a related approach, here involving the monitoring of changes in the fluorescent lifetimes of a single 2-aminopurine (2-AP) base (a fluorescent analogue of adenine) site-specifically substituted in the template strand at the P/T junction, to make similar fractional occupancy measurements. However, we note that structural interpretations of these fluorescence experiments relied heavily on the available crystal structures, and it remained to be shown directly that the 3′-end of the primer in P/T DNA constructs assumes the same distribution of conformations when bound to the protein in solution.To get around this problem, as well as to directly investigate the conformations of the primer DNA in both active sites of the Klenow and Klentaq polymerases, we have used a novel CD spectroscopic approach to characterize the solution conformations of primer DNA bound to Klenow and Klentaq DNAPs. Previously, we had shown that CD spectroscopy, in conjunction with fluorescence measurements, can be used to examine changes in local DNA and RNA conformations at 2-AP dimer probes inserted at specified positions within the nucleic acid frameworks of a variety of macromolecular machines functioning in solution (14Datta K. Johnson N.P. von Hippel P.H. J. Mol. Biol. 2006; 360: 800-813Crossref PubMed Scopus (26) Google Scholar, 15Datta K. von Hippel P.H. J. Biol. Chem. 2008; 283: 3537-3549Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 16Johnson N.P. Baase W.A. von Hippel P.H. J. Biol. Chem. 2005; 280: 32177-32183Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 2-AP is a structural isomer of adenine that forms base pairs with thymine in DNA (and uridine in RNA), and the substitution of 2-AP for adenine in such bp does not significantly perturb the structure or stability of the resultant double helix. Furthermore, when these probes are used as dimer pairs, the CD spectrum primarily reflects the interaction of the transition dipoles of the two probes themselves and thus the local conformation of the DNA at those positions within the P/T DNA. The characteristic CD and fluorescence signals for 2-AP probes in nucleic acids occur at wavelengths of >300 nm, a spectral region in which the protein and the canonical nucleic acid components of the “macromolecular machines of gene expression” are otherwise transparent. In this study, we have examined the binding of Klenow and Klentaq polymerases to P/T DNA constructs that were designed to be comparable with the nucleic acid components of functioning replication complexes. By examining the low energy CD spectra of site-specifically placed 2-AP probes, we have been able to characterize base conformations at defined positions within the DNA to reveal conformational features of specific DNA bases bound at and near both the pol and the exo active sites of these polymerases. These measurements, in that they directly reflect the actual conformations of the DNA chains bound within the active sites of the functioning polymerase, have also provided a direct means to estimate the equilibrium distributions of primer ends between the two active sites for various P/T DNA constructs.DISCUSSIONWe have used the low energy CD and fluorescence spectra of 2-AP probes placed site-specifically in various DNA constructs to determine the local conformations of bases at and near the 3′-primer end of the various DNA structural motifs that can act as substrates for DNA polymerases. Using this technique we have (i) defined the structures of primer and P/T DNA bound at the pol and exo sites of Klenow polymerase, (ii) characterized the conformations of DNA bases bound at defined positions within the active sites, and (iii) determined the relative partitioning of the 3′-primer ends between the pol and exo sites of Klenow for various DNA constructs. Our results also shed light on the role of divalent metal ions in regulating DNA binding and exonuclease activity and in controlling the distribution of the primer termini between the active sites.In some experiments, we used Ca2+ (instead of the canonical Mg2+) as the divalent metal ion cofactor to eliminate the residual exonuclease activity observed in the presence of Mg2+, since low levels of exo activity have been reported for the exo− mutant examined here (17Derbyshire V. Freemont P.S. Sanderson M.R. Beese L. Friedman J.M. Joyce C.M. Steitz T.A. Science. 1988; 240: 199-201Crossref PubMed Scopus (301) Google Scholar). Wherever we could use Mg2+, we have shown that Mg2+ and Ca2+ behave essentially the same in controlling the binding of DNA constructs to Klenow. Substitution of Ca2+ for Mg2+ has previously been used to prevent exonuclease reactions and to trap ternary complexes in structural studies of RB69 DNAP complexes (22Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 39Shamoo Y. Steitz T.A. Cell. 1999; 99: 155-166Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Also, an earlier and a very recent study with Klenow has shown that both binary and ternary complexes can form in the presence of Ca2+ but that the DNA synthesis is significantly inhibited in Ca2+-containing buffers (23Joyce C.M. Potapova O. Delucia A.M. Huang X. Basu V.P. Grindley N.D. Biochemistry. 2008; 47: 6103-6116Crossref PubMed Scopus (101) Google Scholar, 35Gangurde R. Modak M.J. Biochemistry. 2002; 41: 14552-14559Crossref PubMed Scopus (14) Google Scholar). Thus, we conclude that the conformations of the DNA constructs bound in the various complexes studied here probably do represent the physiologically relevant states defined above.Probing Local Conformations at Defined Positions in the Primer-Template DNABased on structural studies, the DNA binding sites of the “large fragment” of DNAP I (Klenow polymerase) can be identified as (i) an exo site that binds ssDNA, (ii) a pol site that binds duplex P/T DNA, (iii) an extended “cleft” that lies between the exo and the pol sites and “clamps” (at somewhat different distances from the primer end) the dsDNA region located just upstream of the P/T junction when the 3′-end of the primer is bound in either the exo or the pol site, and (iv) a binding site that interacts with the single-stranded template downstream of the P/T junction. The protein residues interacting with the DNA in these sites are clearly different for the pol and exo mode-bound complexes (36Kukreti P. Singh K. Ketkar A. Modak M.J. J. Biol. Chem. 2008; 283: 17979-17990Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 40Turner Jr., R.M. Grindley N.D. Joyce C.M. Biochemistry. 2003; 42: 2373-2385Crossref PubMed Scopus (43) Google Scholar). In this study of polymerase-DNA complexes in solution, we have characterized the local conformations of bases interacting with the first three polymerase binding sites listed above. Elsewhere, we have used a different spectroscopic probe to characterize binding site (iv) as well. 4K. Datta et al., manuscript in preparation. Our results with various mismatched P/T constructs binding to Klenow polymerase show that three unpaired nucleotide residues at the 3′-primer end are optimal for primer binding at the exo site. Consistent with earlier crystallographic findings (Fig. 7), we have shown that in solution the 3′-terminal residues of the primer strand of free ssDNA or mismatched P/T junctions adopt a significantly unstacked conformation when bound to the exo site. We have also shown that the P/T DNA duplex adopts an A-like conformation when bound to Klenow at the pol site but not when bound to Klentaq. A B-form to A-form transition for the 2–4 bp of duplex DNA adjacent to the P/T junction upon binding of this DNA region to the pol site has been reported for most DNAP I co-crystals (6Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 7Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 8Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar). It is not clear why such a polymerase-induced conformational change does not occur in equivalent DNA constructs bound to Klentaq, but perhaps this may reflect the weaker binding of our DNA constructs to the latter polymerase, especially for complexes that are free in solution (i.e. not subject to the additional stabilizing interactions that may be present in crystals).We also used our spectroscopic methods to probe the conformation of dsDNA portions of the bound constructs located further upstream of the P/T junction. For Klenow complexes with DNA constructs with a GC bp at the P/T junction (e.g. the P14-1 construct), we observed an A-like conformation for probes located at positions 2 bp upstream from the junction, indicating that these residues are probably bound to a pol site. For Klenow complexed with matched P/T DNA containing an AT-rich junction (the P16-2 construct) and 2-AP probes located further upstream (4 bp) from the P/T junction, the spectra also showed a stacked conformation at the site of the probes. However, this latter conformation deviated significantly from B-form, and most likely reflects a weighted average of conformations at these upstream dsDNA binding sites involved in holding the duplex DNA when the primer termini are partitioned between the pol and the exo binding modes. In contrast, for Klentaq (which contains no exo site) complexed with the P16-2 construct, the 2-AP probes bound in this region exhibited a much smaller divergence from the canonical B-form. Consistent with these findings, structural studies have suggested that when the primer end is in the exo site of Klenow, this duplex DNA sequence is significantly distorted and bent (4Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar) relative to the more B-form-like conformation observed in the same region when the primer is bound in the pol site (8Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar).Probing of the dsDNA sequences at the P/T junction for pol and exo mode complexes with Klenow showed that this region of the DNA construct also adopts distinctly different conformations for the two binding modes. When the primer terminus is bound at the pol site (0mm-A construct in EDTA; Fig. 6B), the dsDNA at the P/T junction takes up a DNA A-like conformation, whereas the duplex P/T junction maintains a B-form structure when the primer end is bound at the exo site (P20-1 construct; Fig. 4B). We speculate that such A-form to B-form conversion of the dsDNA at the P/T junction could serve a mechanistic role that facilitates proper shuttling of the primer terminus between the two active sites in the DNAP I family of polymerases.Partitioning of the Primer between the Two Active Sites of KlenowFor several of the constructs examined in this study, the CD spectra suggested that the 3′-primer ends are partitioned between the pol and the exo active sites. Such partitioning is expected to depend on the sequence and extent of mispairing at the P/T junction region of the DNA constructs. Previous studies of Klenow complexes with DNA, using time-resolved fluorescence anisotropy and a fluorescent probe attached to a base located 8 bp from the P/T junction, had suggested that the presence of 3–4 mismatched bases at the 3′-end of the primer strand at the P/T junction resulted in pushing the equilibrium distribution of the primer >80% toward binding to the exo site, whereas the primer strand of matched P/T DNA constructs seemed to be bound primarily (>85%) at the pol site (12Bailey M.F. Thompson E.H. Millar D.P. Methods. 2001; 25: 62-77Crossref PubMed Scopus (27) Google Scholar, 41Thompson E.H. Bailey M.F. van der Schans E.J. Joyce C.M. Millar D.P. Biochemistry. 2002; 41: 713-722Crossref PubMed Scopus (31) Google Scholar). In our study, we have used similar matched and mismatched DNA constructs but with fluorescent nucleotide analogue probes within the primer strand that interact directly with the relevant binding sites to permit us to identify and directly measure the DNA bound in the two active sites.We have demonstrated that the primer terminus of P/T DNA carrying three mismatched residues at the 3′-primer end binds mostly at the exo site, consistent with previous measurements (12Bailey M.F. Thompson E.H. Millar D.P. Methods. 2001; 25: 62-77Crossref PubMed Scopus (27) Google Scholar). However, in contrast to this earlier study, in which the presence of four terminal mismatches seemed to shift the equilibrium further toward the exo site, our results with 2-AP probes and CD spectra suggest that more than three terminal mismatched base pairs at the P/T DNA junction result in a primer end that is too extended to fit properly into the Klenow exo site. This difference may reflect the fact that the previous workers (12Bailey M.F. Thompson E.H. Millar D.P. Methods. 2001; 25: 62-77Crossref PubMed Scopus (27) Google Scholar) relied on detecting apparent differences in the extent of burial within the protein of a fluorophore attached at a dsDNA site further removed from the P/T junction when the 3′-primer terminus was bound either in the pol or in the exo site of Klenow. Thus, these studies could not directly monitor the conformations of the primer strand bound at the two sites nor conclusively establish a linear relation between binding and probe fluorescence intensity. Nevertheless, our quantitative results (see below) are in general accord with the conclusions of these earlier workers. Our results also indicate that in the absence of divalent metal ions, the primer terminus of a matched P/T DNA is bound to the pol site of Klenow in duplex form.Using CD spectra obtained with the 3mm construct in Ca2+ and the 0mm-A construct in EDTA to serve as reference end points for complete exo- and pol site binding, respectively, we deconvoluted the CD spectra obtained with the Klenow-bound 1mm and 0mm-A constructs to obtain the distribution of the primer ends into the respective active sites. On this basis, we calculated an ∼43% occupancy of the exo site by the 3′-primer end for the 0mm-A construct in Ca2+ buffer, indicating that Klenow can efficiently unwind the fully matched P/T DNA duplex in these constructs. We note here that the three terminal base pairs at the P/T junction of this construct were 5′-C(2-AP)(2-AP)-3′ (i.e. two AT-pairs at the 3′ terminus; see Fig. 1). In contrast, for the primer DNA of the fully matched constructs used by Millar and co-workers (41Thompson E.H. Bailey M.F. van der Schans E.J. Joyce C.M. Millar D.P. Biochemistry. 2002; 41: 713-722Crossref PubMed Scopus (31) Google Scholar, 42Carver Jr., T.E. Hochstrasser R.A. Millar D.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 10670-10674Crossref PubMed Scopus (72) Google Scholar), where the corresponding bases were 5′-ATG-3′ (a GC pair at the 3′ terminus), they estimated ∼14% of the primer end to be at the exo site. Making their P/T junction more GC-rich (5′-GGG-3′) reduced the apparent partitioning into the exo site to ∼7%. Taken together, these results are consistent with the notion that the positional distribution of the primer end between the two active sites is strongly dependent on the thermodynamic stability of the base pairs at the P/T junction and on the efficiency with which the polymerase involved unwinds the P/T junction.In this context, we note that the CD (and fluorescence) results for Klenow binding to our P14-1-matched construct with a GC-rich P/T junction (5′-(2-AP)CC-3′) suggests that the 2-AP probes located upstream of the two terminal C residues are mostly stacked and base-paired. Although we have not probed the 3′-primer end of this construct, if we assume that ∼3 terminal primer nucleotides are unwound to an extent comparable with that of DNA constructs with AT base pairs at the P/T junction (0mm-A or T constructs), the 2-AP dimer probes in the P14-1 construct would be expected to show some unstacking. However, no significant unstacking of the probes in the P14-1 construct was seen, further supporting the suggestion that the extent of unwinding of the primer strand from matched P/T DNA constructs is dependent on the thermodynamic stability of the P/T junction.We observed that ∼61% of the primer ends of the 1mm construct were bound at the exo site when the three terminal bases in our study were 5′-C(2-AP)(2-AP)-3′ (the underlined base here represents the mismatch). In contrast, the 1mm constructs used by Millar and co-workers (41Thompson E.H. Bailey M.F. van der Schans E.J. Joyce C.M. Millar D.P. Biochemistry. 2002; 41: 713-722Crossref PubMed Scopus (31) Google Scholar, 42Carver Jr., T.E. Hochstrasser R.A. Millar D.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 10670-10674Crossref PubMed Scopus (72) Google Scholar) showed exo site distributions of ∼18 and ∼40% for GC-rich (5′-GGG-3′) and AT-rich (5′-ATG-3′) P/T junctions, respectively. Our results agree with the previous observation that the fraction of primer ends located in the exo site increases with the increasing number of mismatches (up to three unpaired nucleotides). However, we note that the actual fraction of primer ends in the exo site in our studies is significantly higher than reported previously. Our results, showing a higher partitioning of the primer terminus into the exo site for matched DNA, are consistent with structural studies of Klenow and another proofreading-proficient polymerase (RB69 from the pol-α family). In these co-crystals, involving fully matched P/T DNA, the primer end exhibited an exo site-bound conformation (4Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar, 39Shamoo Y. Steitz T.A. Cell. 1999; 99: 155-166Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), suggesting this to be the favored equilibrium primer position for a matched P/T DNA construct bound to these polymerases.The Roles of Divalent Metal Ions in DNA Binding and Partitioning of the Primer Terminus between the Two Active Sites in KlenowThe involvement of divalent metal ion co-factors in the catalytic activities of DNAP I polymerases have been extensively characterized. It is well established that two divalent metal ions, coordinated by amino acid residues and the DNA substrate, are essential for the 3′ → 5′ exonuclease activity of Klenow (43Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (914) Google Scholar). Based on structural data from other homologous polymerases, phosphodiester bond formation at the pol site is also believed to involve a two-metal ion mechanism (44Brautigam C.A. Steitz T.A. Curr. Opin. Struct. Biol. 1998; 8: 54-63Crossref PubMed Scopus (334) Google Scholar, 45Johnso
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