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• W2155571717 abstract "Kinetics of human polymerase β binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ∼600–1200 s−1. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO4− group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap. Kinetics of human polymerase β binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ∼600–1200 s−1. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO4− group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap. Polymerase β is one of a number of recognized DNA-directed polymerases of the eukaryotic nucleus (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 2Budd M.E. Campbell J.L. Mutat. Res. 1997; 384: 157-167Crossref PubMed Scopus (53) Google Scholar, 3Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar, 4Hubscher U. Nasheuer H.-P. Syvaoja J.E. Trends Biochem. Sci. 2000; 25: 143-147Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). A characteristic feature of human pol β 1The abbreviations used are: pol βpolymerase βssDNAsingle-stranded DNAdsDNAdouble-stranded DNA1The abbreviations used are: pol βpolymerase βssDNAsingle-stranded DNAdsDNAdouble-stranded DNA is a “simplified” set of activities. The enzyme lacks intrinsic accessory activities, such as 3′ or 5′ exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 6Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 7Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1991; 11: 4441-4447Crossref PubMed Scopus (47) Google Scholar). This limited repertoire of activities reflects the very specialized function of the enzyme in human cell repair machinery which includes the gap filling synthesis involved in mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 4Hubscher U. Nasheuer H.-P. Syvaoja J.E. Trends Biochem. Sci. 2000; 25: 143-147Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 6Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 7Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1991; 11: 4441-4447Crossref PubMed Scopus (47) Google Scholar, 8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar, 9Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar, 10Hammond R.A. McClung J.K. Miller M.R. Biochemistry. 1990; 29: 286-291Crossref PubMed Scopus (56) Google Scholar, 11Hoffman J.S. Pillaire M.J. Maga G. Podust V. Hubscher U. Villani G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5356-5360Crossref PubMed Scopus (138) Google Scholar, 12Masumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). Human pol β is a single polypeptide of ∼39,000 kDa. The crystallographic structures of both rat and human pol β have been determined at 3.6- and 2.3-Å resolution (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 14Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (398) Google Scholar, 15Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12762-12777Crossref PubMed Scopus (176) Google Scholar). The feature that distinguishes the pol β structure from other polymerases is the presence of a small 8-kDa domain connected with the tip of the fingers through a tether of 14 amino acids (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 16Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar). The domain possesses the enzymatic ability to catalyze the release of the 5′-terminal deoxyribose phosphate residue from the incised apurinic-apirimidinic site that is a common intermediate product in base excision repair (12Masumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). The active site of the DNA synthesis resides predominantly in the large 31-kDa domain of the enzyme (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 14Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (398) Google Scholar, 15Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12762-12777Crossref PubMed Scopus (176) Google Scholar).A puzzling problem in the DNA recognition mechanism, by a DNA repair polymerase, is the fact that the enzyme must recognize the damaged DNA, containing a small ssDNA gap, in the context of the large excess of the dsDNA. Although the catalytic properties of the domains are established, the role of both domains in the recognition of the DNA substrates is just now emerging (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Jezewska M.J. Rajendran S. Bujalowski W. J. Mol. Biol. 1998; 284: 1113-1131Crossref PubMed Scopus (36) Google Scholar, 19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). Quantitative equilibrium studies have shown that both human and rat pol β bind the ssDNA in two binding modes (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). The binding modes differ in the number of occluded nucleotide residues and have been referred to as the (pol β)16 and (pol β)5 binding modes (19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar, 21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar, 22Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 3295-3307Crossref PubMed Scopus (28) Google Scholar, 23Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2994) Google Scholar). The existence of the two binding modes is a consequence of the presence of the two structural domains of the protein possessing the DNA-binding subsites, with different DNA binding capabilities (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). Both DNA-binding subsites form the total DNA-binding site of the polymerase. In the (pol β)16 binding mode, both the 8- and 31-kDa domains are involved in interactions with the ssDNA, i.e. the total DNA-binding site is engaged in interactions with the nucleic acid. In the (pol β)5 binding mode, only the 8-kDa domain is engaged in interactions with the DNA (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). The subsite located on the 8-kDa domain has similar affinity for both ss and dsDNA, while the subsite on the 31-kDa domain seems to have a preference for the dsDNA, although this has not been rigorously proven.The mechanism of the formation of the (pol β)16 and (pol β)5 binding modes, by human pol β, is a complex, multiple-step sequential process (21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar). In both ssDNA-binding modes the formation of the protein-nucleic acid complex is initiated through the DNA-binding subsite located on the 8-kDa domain (21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar). Thus the DNA-binding subsite on the 8-kDa domain plays the role of the initiation-binding site of the enzyme to the ssDNA. Analysis of the kinetic data revealed that transitions to subsequent intermediates are also generated through interactions at the 8-kDa domain-DNA interface resulting in the engagement of the 31-kDa domain in interactions with the nucleic acid. The DNA-binding initiation role of the subsite located on the 8-kDa domain is reflected in its energetically homogeneous structure and the capability of accommodating DNA oligomers of different lengths with similar affinity (22Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 3295-3307Crossref PubMed Scopus (28) Google Scholar).In the base-excision repair processes, human pol β fills the ssDNA gaps formed in the damaged DNA (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 9Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). Thus, the physiological substrates for the enzyme are gapped DNAs that have a stretch of ssDNA embedded between the primer and the dsDNA downstream from the primer. Thermodynamic studies of human pol β binding to the gapped DNA substrates indicate that the ability of the 8-kDa domain DNA-binding site to interact with different nucleic acid conformations is crucial for anchoring the enzyme on the gap (19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). In these complexes, the 8-kDa domain binds to the ss and/or dsDNA part of the DNA, downstream from the primer, depending on the length of the ssDNA gap. The 31-kDa domain of the enzyme associates with the dsDNA part of the gapped DNA substrate that contains the primer. Thus, similar to the formation of the (pol β)16 binding mode, engagement of the entire total DNA-binding site of the enzyme provides a large increase of the affinity for the specific recognition of the gapped DNA structure.Understanding the mechanistic aspects of the pol β-gapped DNA recognition process is of great importance for elucidation of the polymerase mechanism at the molecular level. Such analysis will also provide important insights into the recognition mechanisms of specific DNA substrates by other nucleic acid-dependent polymerases. The fundamental questions that arise here are the following. How does the mechanism of the gapped DNA recognition differ from the mechanisms of the enzyme binding to the ssDNA in its different binding modes? What is the formation rate of the different intermediates? What are the energetics of the conversions between the different intermediates? How does the length of the ssDNA gap and the presence of the 5′-terminal phosphate group affect the mechanism? Is there a particular step that makes a dominant contribution to the recognition process? Despite its paramount importance, the direct analysis of the dynamics of the gapped DNA substrate recognition by human pol β has not been quantitatively addressed before.In this article, we report the stopped-flow kinetic analyses of human pol β interactions with the gapped DNA substrates that differ by the number of the nucleotide residues in the ssDNA gap. We provide direct evidence that the mechanism of the specific gap complex formation by human pol β is a three-step, sequential reaction. The bimolecular step includes a very fast association with the DNA, through the 8-kDa domain, followed by two docking steps. The dynamics of the bimolecular step is independent of the length of the ssDNA gap. The internal transition, directly following the bimolecular step, is the major docking step, and is characterized by a large, favorable free energy change for the examined gapped DNA substrates. In this step the DNA-binding subsite located on the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO4− group downstream from the primer does not guide the polymerase to the gap, although it stabilizes the first intermediate in the case of the gapped DNA substrate with a longer gap. The data indicate that the phosphate group affects the enzyme orientation in the complex with the DNA and the structure of the ssDNA gap in the complex with the enzyme. Polymerase β is one of a number of recognized DNA-directed polymerases of the eukaryotic nucleus (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 2Budd M.E. Campbell J.L. Mutat. Res. 1997; 384: 157-167Crossref PubMed Scopus (53) Google Scholar, 3Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar, 4Hubscher U. Nasheuer H.-P. Syvaoja J.E. Trends Biochem. Sci. 2000; 25: 143-147Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). A characteristic feature of human pol β 1The abbreviations used are: pol βpolymerase βssDNAsingle-stranded DNAdsDNAdouble-stranded DNA1The abbreviations used are: pol βpolymerase βssDNAsingle-stranded DNAdsDNAdouble-stranded DNA is a “simplified” set of activities. The enzyme lacks intrinsic accessory activities, such as 3′ or 5′ exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 6Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 7Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1991; 11: 4441-4447Crossref PubMed Scopus (47) Google Scholar). This limited repertoire of activities reflects the very specialized function of the enzyme in human cell repair machinery which includes the gap filling synthesis involved in mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 4Hubscher U. Nasheuer H.-P. Syvaoja J.E. Trends Biochem. Sci. 2000; 25: 143-147Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 6Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 7Matsumoto Y. Bogenhagen D.F. Mol. Cell. Biol. 1991; 11: 4441-4447Crossref PubMed Scopus (47) Google Scholar, 8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar, 9Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar, 10Hammond R.A. McClung J.K. Miller M.R. Biochemistry. 1990; 29: 286-291Crossref PubMed Scopus (56) Google Scholar, 11Hoffman J.S. Pillaire M.J. Maga G. Podust V. Hubscher U. Villani G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5356-5360Crossref PubMed Scopus (138) Google Scholar, 12Masumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). Human pol β is a single polypeptide of ∼39,000 kDa. The crystallographic structures of both rat and human pol β have been determined at 3.6- and 2.3-Å resolution (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 14Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (398) Google Scholar, 15Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12762-12777Crossref PubMed Scopus (176) Google Scholar). The feature that distinguishes the pol β structure from other polymerases is the presence of a small 8-kDa domain connected with the tip of the fingers through a tether of 14 amino acids (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 16Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (569) Google Scholar). The domain possesses the enzymatic ability to catalyze the release of the 5′-terminal deoxyribose phosphate residue from the incised apurinic-apirimidinic site that is a common intermediate product in base excision repair (12Masumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). The active site of the DNA synthesis resides predominantly in the large 31-kDa domain of the enzyme (13Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Crossref PubMed Scopus (756) Google Scholar, 14Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1930-1935Crossref PubMed Scopus (398) Google Scholar, 15Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12762-12777Crossref PubMed Scopus (176) Google Scholar). polymerase β single-stranded DNA double-stranded DNA polymerase β single-stranded DNA double-stranded DNA A puzzling problem in the DNA recognition mechanism, by a DNA repair polymerase, is the fact that the enzyme must recognize the damaged DNA, containing a small ssDNA gap, in the context of the large excess of the dsDNA. Although the catalytic properties of the domains are established, the role of both domains in the recognition of the DNA substrates is just now emerging (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Jezewska M.J. Rajendran S. Bujalowski W. J. Mol. Biol. 1998; 284: 1113-1131Crossref PubMed Scopus (36) Google Scholar, 19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). Quantitative equilibrium studies have shown that both human and rat pol β bind the ssDNA in two binding modes (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). The binding modes differ in the number of occluded nucleotide residues and have been referred to as the (pol β)16 and (pol β)5 binding modes (19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar, 21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar, 22Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 3295-3307Crossref PubMed Scopus (28) Google Scholar, 23Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2994) Google Scholar). The existence of the two binding modes is a consequence of the presence of the two structural domains of the protein possessing the DNA-binding subsites, with different DNA binding capabilities (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). Both DNA-binding subsites form the total DNA-binding site of the polymerase. In the (pol β)16 binding mode, both the 8- and 31-kDa domains are involved in interactions with the ssDNA, i.e. the total DNA-binding site is engaged in interactions with the nucleic acid. In the (pol β)5 binding mode, only the 8-kDa domain is engaged in interactions with the DNA (17Rajendran S. Jezewska M.J. Bujalowski W. J. Biol. Chem. 1998; 273: 31021-31031Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). The subsite located on the 8-kDa domain has similar affinity for both ss and dsDNA, while the subsite on the 31-kDa domain seems to have a preference for the dsDNA, although this has not been rigorously proven. The mechanism of the formation of the (pol β)16 and (pol β)5 binding modes, by human pol β, is a complex, multiple-step sequential process (21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar). In both ssDNA-binding modes the formation of the protein-nucleic acid complex is initiated through the DNA-binding subsite located on the 8-kDa domain (21Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 11794-11810Crossref PubMed Scopus (27) Google Scholar). Thus the DNA-binding subsite on the 8-kDa domain plays the role of the initiation-binding site of the enzyme to the ssDNA. Analysis of the kinetic data revealed that transitions to subsequent intermediates are also generated through interactions at the 8-kDa domain-DNA interface resulting in the engagement of the 31-kDa domain in interactions with the nucleic acid. The DNA-binding initiation role of the subsite located on the 8-kDa domain is reflected in its energetically homogeneous structure and the capability of accommodating DNA oligomers of different lengths with similar affinity (22Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 2001; 40: 3295-3307Crossref PubMed Scopus (28) Google Scholar). In the base-excision repair processes, human pol β fills the ssDNA gaps formed in the damaged DNA (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington, D. C.1995Google Scholar, 5Sobol R.W. Horton J.K. Kühn R. Hua G. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 9Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Abstract Full Text PDF PubMed Google Scholar). Thus, the physiological substrates for the enzyme are gapped DNAs that have a stretch of ssDNA embedded between the primer and the dsDNA downstream from the primer. Thermodynamic studies of human pol β binding to the gapped DNA substrates indicate that the ability of the 8-kDa domain DNA-binding site to interact with different nucleic acid conformations is crucial for anchoring the enzyme on the gap (19Jezewska M.J. Rajendran S. Bujalowski W. J. Biol. Chem. 2001; 276: 16123-16136Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 20Rajendran S. Jezewska M.J. Bujalowski W. J. Mol. Biol. 2001; 308: 477-500Crossref PubMed Scopus (27) Google Scholar). In these complexes, the 8-kDa domain binds to the ss and/or dsDNA part of the DNA, downstream from the primer, depending on the length of the ssDNA gap. The 31-kDa domain of the enzyme associates with the dsDNA part of the gapped DNA substrate that contains the primer. Thus, similar to the formation of the (pol β)16 binding mode, engagement of the entire total DNA-binding site of the enzyme provides a large increase of the affinity for the specific recognition of the gapped DNA structure. Understanding the mechanistic aspects of the pol β-gapped DNA recognition process is of great importance for elucidation of the polymerase mechanism at the molecular level. Such analysis will also provide important insights into the recognition mechanisms of specific DNA substrates by other nucleic acid-dependent polymerases. The fundamental questions that arise here are the following. How does the mechanism of the gapped DNA recognition differ from the mechanisms of the enzyme binding to the ssDNA in its different binding modes? What is the formation rate of the different intermediates? What are the energetics of the conversions between the different intermediates? How does the length of the ssDNA gap and the presence of the 5′-terminal phosphate group affect the mechanism? Is there a particular step that makes a dominant contribution to the recognition process? Despite its paramount importance, the direct analysis of the dynamics of the gapped DNA substrate recognition by human pol β has not been quantitatively addressed before. In this article, we report the stopped-flow kinetic analyses of human pol β interactions with the gapped DNA substrates that differ by the number of the nucleotide residues in the ssDNA gap. We provide direct evidence that the mechanism of the specific gap complex formation by human pol β is a three-step, sequential reaction. The bimolecular step includes a very fast association with the DNA, through the 8-kDa domain, followed by two docking steps. The dynamics of the bimolecular step is independent of the length of the ssDNA gap. The internal transition, directly following the bimolecular step, is the major docking step, and is characterized by a large, favorable free energy change for the examined gapped DNA substrates. In this step the DNA-binding subsite located on the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO4− group downstream from the primer does not guide the polymerase to the gap, although it stabilizes the first intermediate in the case of the gapped DNA substrate with a longer gap. The data indicate that the phosphate group affects the enzyme orientation in the complex with the DNA and the structure of the ssDNA gap in the complex with the enzyme. We thank Gloria Drennan Bellard for help in preparing the manuscript." @default.
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• W2155571717 title "Dynamics of Gapped DNA Recognition by Human Polymerase β" @default.
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• W2155571717 doi "https://doi.org/10.1074/jbc.m200918200" @default.
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