Binding of DNA to Large Fragment of DNA ...

Journal of Biomolecular Structure and Dynamics

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20

Binding of DNA to Large Fragment of DNA Polymerase I: Identification of Strong and Weak Electrostatic Forces and their Biological Implications P. N.S. Yadav , J. S. Yadav & M. J. Modak To cite this article: P. N.S. Yadav , J. S. Yadav & M. J. Modak (1992) Binding of DNA to Large Fragment of DNA Polymerase I: Identification of Strong and Weak Electrostatic Forces and their Biological Implications, Journal of Biomolecular Structure and Dynamics, 10:2, 311-316, DOI: 10.1080/07391102.1992.10508649 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10508649

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Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 10, Issue Number 2 (1992), .,Adenine Press (1992).

Binding of DNA to Large Fragment of DNA Polymerase 1: Identification of Strong and Weak Electrostatic Forces and their Biological Implications P.N.S. Yadav1, J.S. Yada~ and M.J. Modak 1* 1

Department of Biochemist?: and Molecular Biology New Jersey Medical School and Academic Computing Center University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103 USA Abstract Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E. coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions. The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pairofvicinal side chains of acidic and basic amino acids. The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA

Introduction DNA polymerase I (pol I) is a multifunctional enzyme that catalyzes the synthesis of DNA during the replication and repair process (1). A large C-terminal fragment ofthis enzyme, known as the Klenow fragment, contains the polymerase active center where the catalysis of the template directed extension of primer DNA strand occurs. In addition, it also retains a 3'-5' exonuclease activity, which removes the mismatched nucleotide that may have been erroneously incorporated into the product (2). The x-ray crystal structure of the Klenow fragment (3) has shown that the polymerase and exonuclease activities reside in two separate domains. The polymerase domain contains the sites for the binding of DNA template-primer and deoxynucleoside triphosphate (dNTP) in a metal chelate form. In order to understand the molecular mechanism of substrate selection as dictated by the template nucleotide, we have initiated a 3D modeling study of the Klenow fragment and have proposed a pre-polymerization ternary complex consisting of enzyme, template-primer and dNTP (4,5). The model is based on the C-alpha coordinates of the Klenow fragment (3). The substrates were appropriately oriented/ *Author to whom correspondence should be addressed.

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Table I Distribution of charged amino acid residues of Klenow fragment around the bound template-primer Major groove Arg-687:Lys-635 Arg-690:Lys-643 Glu-684 His-928 Asp-705

Minor groove Arg-7 55:Glu-752 Arg-84l:Glu-840 His734 Arg-631 Asp-732

Template nucleotide* Glu-883 Glu-710 Asp-882 His-928

Only the charged residues falling within 5.5 A around the DNA are considered. *Residues in the vicinity of the template nucleotide, immediately following the double stranded region, are considered here.

accomodated in the enzyme cavity using specific amino acid residues known to be essential for activity. The model appears to satisfY many biochemical and genetic data concerning the binding of the DNA and dNTP substrates and/or catalytic requirements of the polymerization reaction (5). Furthermore, good agreement between the electrostatic potential observed with our model-built enzyme protein and the one reported using the crystal coordinates of the Klenow fragment has been noted (5,9). The electrostatic potential of enzyme proteins may be viewed as one of the important factors which contributes to the structural stability of the protein as well as its interaction with substrates (6,7). In DNA polymerase reactions, the electrostatic potential around substrate DNA plays a major role in stabilization of enzymeDNA complex (8). We have now carried out the required electrostatic potential calculations on the Klenow fragment of DNA pol I, with its DNA template-primer substrate, in the 3D model that we have proposed. We find that electrostatic potential contours in the DNA-enzyme complex display a distinct pattern which may be important for the facilitation of catalysis of DNA synthesis.

Computational Details The complete structure of the Klenowfragment and the DNA-enzyme complex was generated as described in our earlier work (5). In the present DNA-enzyme complex, only that portion of DNA template-primer which is in contact with the enzyme (i.e. (dA)8-(dT)7) was taken. The charges on protein and nucleic acids were taken from AMBER force field (10). In the electrostatic calculations, an ionic strength of .14M at pH 7.1 was used. The whole complex was mapped onto a grid size of 65X65X65. A dielectric constant of2 was used for the DNA-enzyme complex while 80 was used for the solvent in both systems. The electrostatic potential calculations were carried out using Del Phi (version 2.2.0) (11,12) and the contour maps were displayed using INSIGHT (version 2.0.0). All the computational work was carried out on the IRIS personal work station interfaced with an HP-UNIX main frame.

Figure 1: A stereoscopic view of the electrostatic potential field of the Klenow fragment. Positive field lines are shown in blue, negative in red. The backbone structure of the Klenow fragment is shown in yellow. (reproduced with permission from American Chemical Society, see ref. 5).

Figure 2: A stereoscopic view of the electrostatic potential field of the binary complex consisting of the Klenow fragment and its substrate dAa-dT7 . Positive potential field lines are shown in blue while the negative ones are in red. The substrate structure alone is shown in yellow.

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Results The stereo view of the electrostatic potential contours ofK.lenow fragment (without DNA) is depicted in figure 1. From the figure it is clear that most of the positive field contours are centered in the enzyme cleft region while the negative field contours are spread all over the enzyme surface. This pattern of positive and negative field contours is in accordance with the one reported by Steitz and colleagues using the crystal coordinates of the Klenow fragment (9). Inspection of the figure shows that the positive electrostatic potential fields are largely spread in the proposed DNA binding cleft as well as at the outer surface of the cleft close to the proposed dNTP binding region. The electrostatic potential field of the DNA-enzyme complex is shown in figure 2. There appears to be a relatively strong electropositive potential field and complementary electronegative potential field around the major groove site of DNA Some of the charged amino acid residues which are in the close vicinity of the double stranded DNA and which are capable of interacting with the major and minor groove sites of the DNA, are listed in Table I. The residues that would preferentially interact with template nucleotides, i.e. 2 nucleotides following the double stranded region of the template strand, are also listed in Table I. In the major groove site 5 basic amino acid residues, namely Arg-687, Lys-635, Arg-690, Lys-643 and His-928 and 2 acidic amino acids, namely Glu-684 and Asp-705 are present, while in the minor groove sites, 4 basic amino acid residues, namely Arg-755, Arg-841, His-734 and Arg-631 and 3 acidic amino acids, namely Glu-752, Glu-840 and Asp-732 are present (Figure 3). When the locations of the above residues are examined, it is clear that many of them could form the vicinal pairs consisting of either basic amino

Ly:sr635

Arg\687 '' \ \

Clu-7'5~ Arg-155

Figure 3: A stereoscopic view of the amino acid residues within a 5.5 A sphere around duplex DNA (i.e. d.As-dT7) bound in the cleft. DNA is represented in a dot surface. The specific locations of some of the residues is indicated by name and number in the primary amino acid sequence of pol I.

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acids (e.g. Arg-687:Lys-635 and Arg-690:Lys-643) or a combination of a basic and acidic amino acid (e.g. Arg-755:Glu-752 and Arg-84l:Glu-840). These pairs further appear to align along the major and minor groove sites of the DNA template-primer respectively. The residues Glu-883, Glu-710, and His-928 appear to be involved in positioning of the template nucleotide such that the binding of the proper substrate nucleoside triphosphate would follow.

Discussion We have developed a computer assisted molecular model of the Klenow fragment of E. coli DNA polymerase I (5) using the C-alpha coordinates from the crystal struc-

ture reported by Steitz and colleagues (3). The extensive similarity between the electrostatic potential field observed with our model (Figure 1) and that reported for the crystal based structure (9) strongly suggests that our computer assisted model may closely approximate the real structure. Furthermore, extensive biochemical analysis had revealed the identity and location of the important amino acid residues that play a part in the binding of substrate deoxynucleotides and DNA (13-16). Therefore, using this information as a guideline, we were able to fit a double stranded DNA template-primer into the active site cavity of the Klenow fragment (5) without considering the electrostatic complementarities. The electrostatic potential contours of the modeled complex (Figure 2) clearly show the nature of the electrostatic binding between DNA and poll. This observation provides a strong support to the proposed model and some insight into the specific interactions involved in the enzymatic syntheis of DNA As described in the result section, the most exciting observation in the present study

is the presence of pairs of side chains of charged residues at specific locations along the bound DNA In our earlier chemical modification studies of the Klenow fragment, using active-site-directed reagents, we have identified a number of positively charged residues and have implicated them in a specific binding function. For example. with pyridoxal 5' -phosphate, which forms a Schiff base with Lys-7 58 and Lys-735, we noted that the modification of these residues affects the substrate dNTP binding ( 13) and the mode of DNA synthesis, which shifts from processive to distributive (17). With phenylglyoxal as a site directed reagent, which specifically reacts with Arginine residues (18), we have identified Arg-841 as the site of modification which causes the inability of the enzyme to bind to DNA The interaction between DNA and Arg-690 is predicted by Joyce et al. from polA6 mutant studies (19,20). The presence of these charged residues at the surface of the DNA binding cleft is in agreement with our binding model. Since the binding of template-primer to the enzyme is unaffected regardless of the nucleotide sequence, the role of the electrostatic binding force may be considered to be the major component in templateprimer and enzyme binding. Furthermore, the electrostatic potential field interaction is not only influenced by the interacting residues but also by the other charged residues present in their vicinity. In the present study we find that the side chains of two pairs of basic amino acid residues, Arg-687 and Lys-635, and Arg-690 and Lys643 are interacting with the major groove site. This is reflected in a net positive potential field in the major groove site. We consider these residues to constitute a "DNA anchoring site" in the enzyme protein. The combination of side chains of

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basic and acidic amino acid pairs,Arg-755 and Glu-752, and Arg-841 and G1u-840 found near the minor groove sites, produce less positive potential field in the minor groove sites and would bind DNA template-primer somewhat loosely. We consider the region covered by these residues as the "mobile sites" which may be proposed to stabilize the bound DNA into the cleft. In both major and minor groove sites, additional non-paired charged residues are also present (Table I). Of these, the positively charged residues may interact directly with the phosphodiester backbone of the DNA while negatively charged residues mayinteractthrough a divalent metal bridge. The arrangement of strong and weak binding forces in the Klenow enzyme appears to be well suited for the subsequent polymerization reaction when the hydrolysis of dNTP takes place. The release of the metal pyrophosphate may be postulated to occur in the close vicinity of the rnajor binding region ofthe DNA such that the heat energy released during the cleavage of triphosphate would weaken the binding force present at the anchoring site of the enzyme. In the ternary complex, the position of the P-alpha of the dNTP, the cleavage site during polymerization, is located near the major groove site (5). It also appears likely that, in addition to neutralizing the major groove binding interaction, the energy released during the polymerization step may also facilitate the forward movement of the enzyme protein along the DNA template, as required in the polymerization reaction. Similar types of electrostatic interactions between charged amino acid residue pairs and the DNA surface are also observed in the crystal structure of the DNA-EcoRI endonuclease complex by Rosenberg and colleagues (21). Recently, a comparison of the primary amino acid sequence ofE. coli DNA polymerase I with other prokaryotic DNA dependent DNA polymerases has been carried out to determine the conservation of various amino acid residues in the different enzymes (22,23). The purpose of this comparative study was to predict the common motifs and domains among the polymerase class of proteins. Since the three dimensional structure has only been solved for the Klenow fragment (3), it has been used as a prototype polymerase. We therefore compared the positions of charged amino acid residues that we have found around DNA (Table I) among prokaryotic polymerases (22). We find that Arg-631, Lys-635, Arg-690, Asp-705, Glu-7lO,Asp-732 His-734, Asp-882 and Glu-883 are conserved in most polymerases, with the exception of one or two polymerases. Residues Glu-684, Arg-755, Glu-840 and Arg-687 are found in all bacterial polymerases, while only Arg-841 seems to be present in two polymerases. However, it should be pointed out that in other polymerases Arg-841 has been substituted by positively charged residues, i.e. lysine or histidine. Thus an overall similarity in the distribution of charged residues in various prokaryotic polymerases suggests a similarity in the tertiary folding structure as well as the structure of the DNA binding cleft, at least in these polymerases. In eukaryotic and other polymerases, a domain structure which displays a charge distribution similar to the Klenow fragment may occur, however participating residues and their locations in these enzymes have not been clarified.

Acknowledgements We thank Drs. Leslie Michelson and Swamy Laxminarayan for their encouragement

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and for providing us with molecular modeling facilities at the Academic Computing Center of the university. This research was supported by a grant from National Institute of Health(# NIGMS-36307). References and Footnotes 1. A Kornberg and T. Baker, DNA Replication, Freeman and Company, San Francisco (1991). 2. D. Brutlag, M.R. Atkinson, P. Setlow and A Kornberg, Biochem. Biophys. Res. Commun. 37, 982 (1969). 3. D.L. Ollis, P. Brick, R. Hamlin, N.G. Xuong and T.A. Steitz, Nature 313, 762 (1985). 4. J. Yadav, P.N. Yadav, E. Arnold, S. Lakshminarayan and M.J. Modak, in Protein: Structure, Dynamics and Design, Eds., V. Renugopalkrishnan, P.R. Karey,I.C.P. Smith, S.G. Haung and A C. Storer, ESCOM Science Publisher B.V., Leiden, p 330 (1991). 5. P.N.S. Yadav, J.S. Yadav and M.J. Modak, Biochemistry 31,2879 (1992). 6. J.B. Matthew, Ann. Rev. Biophys. Biophys. Chern. 14, 387 (1985). 7. B. Honig, W. Hubbel and R. Flewelling, Ann. Rev. Biophys. Biophys. chem. 15, 163 (1986). 8. B. Jayaram, K.A. Sharp, and B. Honig, Biopolymers 28, 975 (1989). 9. J. Warwicker, D.L. Ollis, F.M. Richards and T.A. Steitz,.J. Mol. Bioi. 186,645 (1985).

10. S.J. Weiner, P.A. Kollman, D.A. Case, U.C. Singh, C. Ghio, G. Alagona, S. Profeta, and P. Weiner, J Am. Chern. Soc. 106, 765 (1984). 11. 12. 13. 14. 15. 16. 17. 18. 19.

I. Klapper, R. Hagstrom, K. Sharp and B. Honig. Proteins 1, 47 (1986). M. Gilson, K. Sharp, and B. Honig,.J. Comp. Chern. 9, 327 (1988). A Basu and M.J. Modak, Biochemistry 26, 1704 (1997). V.N. Pandey and M.J. Modak,J Bioi. Chern. 263,6068 (1988). V.N. Pandey, K.R. Williams, K.L. Stone and M.J. Modak, Biochemistry 26, 7744 (1987). V.N. Pandey, N.A. Kaushik, D.S. Pradhan and M.J. Modak,J Bioi. Chern. 265,3679 (1990). S. Basu, A Basu and M.J. Modak, Biochemistry 27,6710 (1988). P.M. Mohan, A Basu, S. Basu, K.l. Abraham and M.J. Modak. Biochemz:~try 27,226 (1988). C.M. Joyce, D.M. Fujii, H.S. Laks, C.M. Hughes and N.D.F. Grindley, J Mol. Bioi. 186, 283

(1985). 20. W.S. Kelley and N.D.F. Grindley, Nucleic Acid Res. 3, 2971 (1976). 21. J.A McClarin, C.A. Frederick. B. Wang, P. Greene, H.W. Boyer, J. Grable, and J.M. Rosenberg, Science 234, 1526 (1986). 22. M. Delarue, 0. Poch, N. Tordo, D. Moras and P. Argos, Prot. Engineer. 3, 461 (1990). 23. J. Ito and D.K. Braithwaite, Nuc. Acid~ Res. 19.4045 (1991).

Date Received: September 17, 1991

Communicated by the Editor D.L. Beveridge