The Major Difference in the Structures of Klentaq 1 in Comparison to Klenow pol 1 - Research Paper Example

 ABSTRACT Taq polymerase or the DNA polymerase isolated from Thermus. aquatcus, a thermophilus bacterium, is a DNA polymerase I type of enzyme, involved in DNA repair and has the same basic structure as the DNA polymerase I isolated from E. coli. However, the enzyme has some unique characters of its own, specifically one that impart stability at high temperatures; enabling the survival of T. aquaticus in hot springs; and rendering the enzyme suitable for use in biotechnological applications. And therefore; it becomes imperative to study the structure of Taq polymerase individually. INTRODUCTION: Cell proliferation and genomic inheritance are integral steps to growth and development of life form on one hand and to continuation of species on the other hand. Fidelity in copy of genes i.e. replication is the determining criteria in both these events (Stoeber and others, 2001). The enzyme responsible for replication by adding on precise dNTPs with respect to the template DNA strand and proofreading the same is DNA polymerase (Joyce and Steitz, 1994). DNA polymerases are a highly variable family of enzymes having the same primary function of adding dNTPs to primer strand (Steitz, 1999). DNA polymerases find significant uses in analysis of genetic material and cloning, due to efficiency in faithful replication of genetic material. Using PCR (polymerase chain reaction), the number of copies of target gene sequences can be increased manifold (Mullis and Faloona, 1987), thus helping overcome the major limitation of limited availability of target sequences. However; due to the high temperatures required for DNA denaturation, the thermolabile DNA polymerase I of E. coli became a major limitation for PCR (Saiki and others, 1988). This limitation was overcome with Taq polymerase (EC 2.7.7.7), which had been isolated from thermophilic bacteria Thermus aquaticus in 1965 by Thomas D. Brock and established to be stable even at temperatures of 95.C (Brock and Edwards, 1970, Saiki et. al 1988). 1 ISOLATION AND CHARACTERIZATION: Study of the replication mechanism remained relatively obscure in thermophiles till much later. In 1976, Chien and others (table 1 and 2) studied it by isolating and characterizing the polymerase from T. aquaticus, while Lawyer and co workers (1993) studied the same using expression of a Taq DNA polymerase by Taq DNA polymerase 1 gene (taq pol 1), followed by its purification. Table 1: Summary of the purification procedure a (Chien and others, 1976) Fraction Vol (ml) Total polymerase activity (U) Total protein (mg) Yield of Activity (%) SpAct (U/mg) Purification (fold0 Crude 176 2080 975.0 2.13 DEAE-Sephadex 210 4613 197.5 221 23.3 10.9 Phosphocellulose 132 1900 16.0 91 118.7 55.7 DNA-cellulose 63 685 33 Assay Result (Chien and others) Result (Lawyer and others) 5’to 3’Exonuclease Activity negative Positive 3’to 5’Exonuclease Activity negative Negative Temperature optima 80.C 80.C pH optima 7.8 9.4 Divalent cation for optimal activity 10mM Mg 2+ 2mM Monovalent cations Low levels of NaCl (40mM) and KCl (60mM) KCl (55mM) Molecular weight 68000 D (by sucrose gradient centrifugation) 63000 D (by gel filtration on sephadex G100) 94000 D (by SDS polyacrylamide gel electrophoresis) 93900 (based on sequence) Specific Activity(pure enzyme) 292000 a One unit of polymerase equals the incorporation of 10nmol of [3H]dTTP into acid soluble material at 80.C in 30min. Table 2: Taq polymerase characteristics (Lawyer and others, 1993, Chien and others, 1976) 2 STRUCTURE: Taq polymerase belongs to a group of enzymes collectively known as DNA polymerase I, on the basis of their similarity to DNA polymerase I isolated from bacterium E.coli (Kornberg, 1956). It is speculated that the two enzymes share a common ancestry with evolutionary subsequent alterations. The molecule is a monomer, with a molecular weight estimated to be 93.9kDa (Lawyer et al., 1993), and can be clearly differentiated into three domains: Residue 1-290 forming the N-terminal domain with 5’to 3’nuclease activity. Residue 291-419 forming the central domain, without any reported enzymatic activity. Residue 420-832 forming the C-terminal domain. Taq polymerase shows significant similarity to E.coli DNA polymerase I, with the primary sequence exhibiting 38% homology, maximum similarity being in C-terminal primary sequences, the structures of which are also significantly similar (Kim and others, 1995). DNA polymerase I being the first and most studied enzyme of family, most studies on Taq polymerase present comparative accounts of the two enzymes, the former giving a ground to relate to and therefore easily comprehend the latter. A detailed study of Klentaq 1 X ray crystal structure detrmined at 2.5 Å resolution, refined to an R factor of 22.5%, and an average B factor of 25 Å2 post temperature factor refinement; was done by Korolev et al (1995). Klentaq 1 is the larger fragment of Taq polymerase comprising of residues 291 to 832, excluding the N terminal domain. Korolev and co workers crystallized a modified Klentaq 1 carrying a 7 residue N-terminal extension; and structure determination was done using heavy metals Uranyl and platinum compounds. Klentaq 1 and corresponding klenow fragment (residues 516-928) of E.coli polymerase 1 show a 49.6% similarity (figure 1), with similarity obvious in the folds of the two fragments. The three subdomains of Klentaq 1, like Klenow pol 1 show a similarity in structure to the thumb, the palm amd the fingers, with differences in the two apparent only in the fingertip region. The cleft formed by the three subdomains is of optimal shape and size to allow the binding of double stranded B-DNA substrate and the flexibilty of the subdomains warrants that the enzyme envelopes the DNA completely to allow continuous polymerization and replication (Ollis et al, 1985). The major difference in the structures of Klentaq 1 in comparison to Klenow pol 1 are: O1 helix terminates earlier Helices H and I are inclined towards N terminal residues, resulting in a shift in the position of N terminal residues of helix I by approximately 4A. A. The N terminal domain of Klentaq1 is much more compact than that of Klenow pol 1 and merges smoothly into the palm region which then forms a large flat area extending along the entire length of enzyme, the entire structure resembling a wishbone (Korolev, et al., 1995). There are significant similarities with Klenow polymerase 1, despite the fact that it neither posesses exonuclease activity, nor are the sequences of residues in the two similar. Still the strands 1, 2, 3 and 4, and also the helices B, C, D, E and F are similar in the two (figure 1). However, there is also a significant differences in this domain of the two enzymes, the helix A is absent in Klentaq1 and instead is present a proline rich loop. The intervening region between helices E and F, which formed an extensive loop in Klenow pol 1, is also missing in Klentaq1, and the F & B helices too are smaller. Another minor alteration is shortening of loop between strand 4 and helix B by 10 amino acids (Korolev and others, 1995). Also, the distinct feature of Taq polymerase intervening domain rendering it incapable of catalytic activity is that the dTMP binding carboxylate residue containing sites of E. coli are either replaced or missing (Kim et al., 1995). In strand 2 Glu357 and Thr358 are missing, Asp355 is replaced by Phe309 and in helix C Asp424 is replaced by Leu356. Taq polymerase site is thus, hydrophobic and incapable of exonuclease activity(figure 2) (Li and others, 1998). B. The interface between N-terminal and C-terminal domains is formed by the residues belonging to helices C and D, connecting loop of helices D and E of small N-terminal domain, and helix G, strand 7 and interconnecting loop of strands 7 and 8 of large C-treminal domain. Helix C and helix G in Klentaq1 are profusely in contact with each other, running alomost parallel to each other, resulting in a larger hydrophobic core in Klentaq1, compared to Klenow pol 1; and therefore more stability (Korolev and others, 1995). C. The structure of central domains of Klentaq and Klenow fragment are almost identical, both exhibiting polymerase activity. The structure of polymerase site in action, bound to dNTPs has been extensively studied by Li and others (1998) by X ray diffraction at a resolution of 2.5 Å (R factor 22.3) and Eom and others (1996). . D. The 5’ nuclease domain of Taq polymerase was studied by Kim et al (1995) by X ray diffraction at 2.4 A resolution, and was found to consist of conserved group of divalent metal ions binding carboxylate residues at the bottom of cleft. The distance between the catalytic sites of nuclease and polymerase domains is only 70 Å in the crystal studied and thus explains the coordinated mechanism of action of the two domain resulting in the formation of double stranded DNA with only one nick. Urs and others (1999) from study of a different crystal form at 2.6 Å resolution showed that orientation of nuclease domain is different and much closer to polymerase active site, compared to that reported for native enzyme and DNA complexes of enzyme, but identical to that of enzyme Fab complex (Murali and others, 1998). Occurrence of diverse orientations is also indicative of relative mobility between the two active sites of the enzyme in vivo. C-TERMINAL DOMAIN AND DNA POLYMERISATION: Eom et al. (1996) from studies of crystal structure of enzyme bound to DNA duplex and found that the DNA does not passes through the polymerase cleft, instead a protein side chains bind to DNA blunt end through a minor wide groove in the enzyme with polymerase and nuclease binding sites of a part of DNA being partially shared The crystal structure shows that the primer template draws near the polymerase domain from central domain side of cleft; as noted in earlier DNA replication studies. The blunt end of dsDNA is also in close contact with O helix, with the 3’hydroxyl of primer close to catalytic core. The catalytic core is formed by the residues Asp785, Glu786, and Asp610; with the dNTPs binding adjacent to O helix, all four types in binding in similar positions secured by three positively charged amino acid residues viz. Arginine (659), lysine (663) and arginine (587); and two polar residues namely histidine (639) and glutamine (613) (Figure 3). While the triphosphates irrespective of parent molecule, have similar orientations in polymerase-dNTP complexes; the directions of sugars and bases varies with the parent molecule proving that primary dNTP substrate recognition is based on phosphate moiety. The base on the other hand is seen in two specific conformations (Eom and others, 1996 and Li et al, 1998), with only one conformation being compatible with structure imposed as a result of DNA binding. Figure 4: Kinetic model for DNA replication (Rothwell et al., 2005) Conversely, small angle X ray scattering studies of Taq polymerase structure in solution revealed that the global conformation of the enzyme molecule remains unaltered as a consequence of matched primer DNA template or ddATP binding, the radius of gyration 38.3Å remaining constant pre and post binding (Joubert and others, 2003). Also 5’nuclease domain remains extended, resembling more the elongated crystal structure rather than the compact one, though not identical to it; which was quite puzzling considering the coordinated functioning of the sites. However Ho and others (2004) have found using contrast variation solution small angle neutron scattering studies on Taq polymerase bound to “overlap flap” DNA in solution, that while free enzyme has expanded conformation, event of DNA binding makes it compact, with the thumb and palm subdomains of polymerase site coming closer. Thus, though over all mechanism of replication is clear (figure 4), nothing conclusive can be said about the close and open form transformation being the rate determining step of the replication mechanism. For direct visualisation of the mechanism of fluorescence resonance energy transfer technology was developed (Rothwell et al., 2005). It adds a donor and receptor fluorophore to the protein/dNTP complex and thereby helps observe the finger subdomains (figure 5, 6). It was noticed that the closing of this domain was much quicker than the kinetically determined rate limiting step. Thus it has been speculated that the rate limiting step occurs after the complex is formed at the active site. COMPARISON OF Taq POLYMERASE WITH POLYMERASE 1: Besides the structural similarities and differences already discussed, the major difference between the two enzymes is with respect to thermostabiltiy. Thermal denaturation of both is irreversible, the Klenow1 denatures at 37.C while Klentaq1 at 100.C at pH 9.5 (Karantzeni and others, 2003). The molecular basis of thermostabilty of Klentaq1 has been studied in depth by Kremlov and others (1995) and has deduced that amino acid substitutions contribute to the thermostable properties of the enzyme. Of the 79 reported substitutions, most replace charged residues to oppositely charged ones, with only six conserving charge which are either lysine to arginine or vice versa. As a consequence of this charge shuffling and alterations in ion pairing pattern, the stability of the Klentaq 1 is different from that of Klenow pol 1, a precise estimation of which was provided by the calculation of electrostatic component of free energies of folding of the two enzymes, measured by continuum electrostatic method (Gilson and Honig, 1988). It was found that in both N-terminal and the larger domain, the estimates favoured Klentaq 1, with differences being more in prior. The thermodynamic basis of the thermal stability of Taq polymerase 1 and DNA pol1 was studied by Karantzeni and others (2003), using differential scanning colorimetry. Both the enzymes comprise of two separate thermodynamically unfolding domains, the 5’ nuclease and the larger domain. While in Taq polymerase, the 5’nuclease domain denatures 10.C below the Klentaq domain; in Polymerase 1 the two melt simultaneously. Also the kinetic barrier to irreversible denaturation of Klentaq was found to be much higher than that of Klenow, as revealed by denaturation scan rate dependence analysis with Arrhenius formalism. Taq Polymerase and PCR: Polymerase chain reaction or PCR involves exponential amplification of DNA fragments using two oligonucleotide primers flanking the target DNA, by repeated cycles of denaturation, polymerisation and denaturation (Mullis and Faloona, 1987) (figure 4). The major limitation of this procedure was the thermolabilty of Klenow fragment derived from E. coli, since the denaturation step involves high temperatures, which was overcome with use of thermostable Taq polymerase (Saiki and others, 1988). The lack of 3’ to 5’ exonuclease or proof reading ability in Taq polymerase, leads to an error rate of, 1*10-5 errors/base, depending on the reaction conditions (Eckert and Kunkel, 1991); still it is popularly used when high fidelity is not important, since proof reading reduces primer length leading to amplification of non specific sequences. Moreover lack of proof reading leads to template independent addition of a nucleotide to the 3’ end of product known as ‘A overhang’, which simplifies cloning (Zhou and others, 1995). For amplification of longer sequences (>10 kb) , a combination of Taq polymerase and a proof reading polymerase can be used, or conversely a modified version of Taq polymerase with N-terminal removed can also be used. Importance of Taq polymerase led to a number of mutational analyses of the enzyme to improve its performance. Merkens et al. (1995) identified Arg25 and Arg74, while Kim and others (1997) identified Arg74, Lys82, and Arg74, which when replaced by Lysine cause lower 5’nuclease activity, retaining the polymerase activity. Cold sensitive mutants of Taq polymerase which avoid non specific priming, with mutations located at periphery of enzyme, away from the active site (Kermekchiev and others, 2003). A 708 codon mutation in both Klentaq and Taq polymerase rendering enzyme highly resistant to PCR inhibitors in blood and soil samples (Kermekchiev and others, 2009). Triple mutants of Taq polymerase, omni Taq and omni Klentaq which resist inhibition by 20-25% whole blood serum and soil samples (Zhang and others, 2010). CONCLUSION: Taq polymerase is structurally and functionally very similar to DNA polymerase 1 isolated from E. coli, yet the sequence of the protein has some major alterations rendering it unique with its thermostability, which is the chief cause of the special interest in the enzyme. Since its first incorporation in PCR by Saiki and co workers (1988), the enzyme has variously been used, in native as well as mutant forms to achieve desired results in biotechnological applications. Yet, the structure and mechanism of Taq polymerase is not fully understood and further studies are recommended for exploiting the potentials of the enzyme. References 1. Brock, T. D., and Edwards, M. R., “Fine structure of Thermus aquaticus, an extreme thermophile,” J Bacteriol, 104(1970): 509-17. 2. Chien, A., Edgar, D. B., and Trela, J. M., “Deoxyribonucleic acid polymerase from the extreme thermophile Termus aquaticus” J. of Bact, 127(1976): 15550-7. 3. Eckert, K. A, and Kunkel, T. A., “DNA polymerase fidelity and polymerase chain reaction,” PCR methods appl, 1(1991): 17-24. 4. Eom, S. H., Wang, J., and Steitz, T. A.”Structure of Taq polymerase with DNA at the polymerase active site,” Nature, 382(1996): 278-281. 5. Gilson, M. K., and Honig, B., “Calculating the electrostatic potential of molecules in solution,” Proteins, 4(1988), 7-18. 6. Ho, D. L., Byrnes, M., Ma, W., Shi, Y., Callaway, J. E., and Bu, Z., “Structure specific DNA induced conformational changes in Taq polymerase revealed by small angle neutron scattering,” The journal of Biological Chemistry, 279(2004): 39146-54. 7. http://www.virtualmedicalcentre.com 8. Joubert, A. M., Byrd, A. S. and LiCata, V. J., “Global conformations, hydrodynamics, and X-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution,” The journal of Biological Chemistry, 278(2003): 25341-7. 9. Joyce, C. M. and Steitz, T. A., “Function and structure relationships in DNA polymerases,” Annu. Rev. Biochem., 63(1994): 777-822. 10. Kermekchiev, M. B., Kirilova, L. I., Vail, E. E., and Barnes, W. B., “Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples,” Nucleic acids res, 37(2009). 11. Kermekchiev, M. B., Tzekov, A., and Barnes, W. M., “Cold-sensitive mutanys os Taq DNA polymerase provide a hot start forPCR,” Nucleic acids res., 31(2003): 6139-47. 12. Kim, Y., Eom, S. H., Wang, J., Lee, D. S., Suh, S. W., and Steitz, T. A., “Crystal structure of Thermus aquaticus DNA polymerase,” Nature, 376(1995): 612-6. 13. Kornberg A., “Enzymic synthesis of deoxyribonucleic acid,” Biochemica et biophysica acta, (1956): 197-8. 14. Korolev, S., Nayal, M., Barnes, W. M., DiCera, E., and Waksman, G., “Crystal structure of the large fragment of Thermus aquaticus DNA polymerase 1 at 2.5 A resolution: structural basis of thermostability,” Proc. Natl. Acad. Sci., 92(1995): 9264-8. 15. Lawyer, F. C., Stoffel, S. Saiki, R. K., and others, “High level expression, purification, and enzymatic characterization of full length Thermus aquaticus DNA polymerase and a truncated form deficient in 5’ to 3’ exonuclease activity,” Genome Res. 2(1993): 275-87. 16. Li, Y., Kong, Y., Korolev, S., and Waksman, G., “Crystal structure of the Klenow fragment of Thermus aquaticus DNA polymerase 1 complexed with deoxyribonucleoside triphosphates,” Protein Science, 7(1998): 1116-23. 17. Merkens, L. S., Bryan, S. K., and Moses, R. E., “Inactivation of the 5’-3’ exonuclease of Thermus aquaticus DNA polymerase,” Biochem Biophys acta, 1264(1995): 243-8. 18. Mullis, K.B. and Faloona, F.A., Methods Enzymol. 155(1987): 355-350. 19. Murali, R., Sharkey, D. J., Daiss, J. L., and Krishna Murthi, H. M., “Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.,” Proc. Natl. Acad. Sci., 95(1998): 12562-7. 20. Ohmori, H., Friedberg, E. C., Fuchs, R.P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R., “The Y-Family of DNA Polymerases,” Mol Cell, 8(2001): 7-8. 21. Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G. And Steitz, T. A., “Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP,” Nature, 313 (1985): 762-6. 22. Rothwell, P. J., Mitaksov, V., and Waksman, G., ‘Motions of the fingers subdomain of Klentaq1 are fast ans not rate limiting: Implications for the molecular basis of fidelity in DNA polymerases,” Molecular cell, 19(2005):945-55. 23. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, S. T., Mullis, K. B., and Erlich, H. A., “Primer directed enzymatic amplification of DNA with thermostable DNA polymerase,” Science, 239(1998): 487-91. 24. Shadel, G. S. and Clayton, D. A., “Mitochondrial DNA maintenance in vertebrates,” Annu Rev Biochem, 66(1997): 409-435. 25. Sharief , F. S., Vojta, P. J., Ropp, P. A. & Copeland, W. C., “Cloning and chromosomal mapping of the human DNA polymerase theta (POLQ), the eighth human DNA polymerase,” Genomics, 59(1999): 90-96. 26. Steitz, T., “DNA polymerases; structural diversity and common mechanism,” J. Biol. Chem, 274(1999): 17395-8. 27. Stoeber, K., Tisty, T. D. And others, “DNA replication licensing and human cell proliferation,” Journal of cell science, 114(2001): 2027-41. 28. Urs, U. K., Murali, R., and Krishna Murthy, H. M., “Structure of Taq DNA polymerase shows a new orientation for the structure-specific nuclease domain,” Acta Cryst, 55(1999): 1971-77. 29. Zhang, Z., Kermekchiev, M. B., and Barnes, W. M., “Direct DNA amplification from crude clinical smaples using PCR enhancer cocktail and novel mutants of Taq,” J Mol Diagn, 12(2010): 152-61. 30. Zhou, M.Y. and others, “Universal cloning method by TA strategy, Biotechniques 19 (1995): 34–5. Read More