Structure of lithocholic acid binding to the N-terminal 8-kDa domain of DNA polymerase beta

The purpose of this study was to investigate the molecular action of lithocholic acid (LCA), known as a selective inhibitor of DNA polymerase beta (pol beta). The 39-kDa pol beta was separated proteolytically into two fragments of the template-primer binding domain (8 kDa) and the catalytic domain (31 kDa). LCA bound tightly to the 8-kDa fragment but not to the 31-kDa fragment. We examined the structural interaction with the 8-kDa domain using LCA. On (1)H-(15)N HMQC NMR analysis of pol beta with LCA, the 8-kDa domain bound to LCA as a 1:1 complex with a dissociation constant (K(D)) of 1.56 mM. The chemical shifts were observed only in residues mainly in helix-3, helix-4, and the 79-87 turn of the same face. No significant shifts were observed for helix-1, helix-2, and other loops of the 8-kDa domain. This region was composed mainly of three amino acid residues (Lys60, Leu77, and Thr79) of pol beta on the LCA interaction interface. The inhibition mechanism and the structure-function relationship between pol beta and LCA is discussed.     

Mode analysis of a fatty acid molecule binding to the N-terminal 8-kDa domain of DNA polymerase beta. A 1:1 complex and binding surface.

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C(18)-linoleic acid (LA) or C(24)-nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an Omega-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K(D)) of 1.02 or 2.64 mM, respectively.     

Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA binding modes

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.     

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.     

Energetics and specificity of Rat DNA polymerase beta interactions with template-primer and gapped DNA substrates

Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.     

Structural relationship of lithocholic acid derivatives binding to the N-terminal 8-kDa domain of DNA polymerase beta

We reported previously that lithocholic acid (LCA, 3-alpha-hydroxy-5-beta-cholan-24-oic acid), one of the major compounds in the secondary bile acids, selectively inhibited the activity of mammalian DNA polymerase beta (pol beta) [Mizushina, Y., Ohkubo, T., Sugawara, F., and Sakaguchi, K. (2000) Biochemistry 39, 12606-12613]. The purpose of this study was to investigate the molecular structural relationship of LCA and its 10 chemically synthesized derivatives. The inhibitory activities of pol beta by some derivative compounds were stronger than that by LCA, and these compounds bound tightly to the 8-kDa domain fragment but not to the 31-kDa domain fragment of pol beta. Biacore analysis demonstrated that the 8-kDa domain bound selectively to compound 9 (3-alpha-O-lauroyl-5-beta-cholan-24-oic acid), which was the strongest pol beta inhibitor tested, as a 1:1 complex with a dissociation constant (K(d)) of 1.73 nM. From computer modeling analysis (i.e., molecular dynamics analysis), the 8-kDa domain had two inhibitor binding areas. Three amino acid residues (Lys60, Leu77, and Thr79) of the 8-kDa domain bound to LCA and compound 2 (3-alpha-methoxy-5-beta-cholan-24-oic acid), and four amino acid residues (Leu11, Lys35, His51, and Thr79) of the 8-kDa domain bound to compound 9. From these results, the structure-function relationship among pol beta and its selective inhibitors was discussed.     

Rapid segmental and subdomain motions of DNA polymerase beta

DNA polymerase (pol) beta is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between alpha-helix N and the nascent base pair. Kinetic and structural characterization of pol beta has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and alpha-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of alpha-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol beta were determined. The results revealed that the segmental motion of alpha-helix N was rapid ( approximately 1 ns) and far more rapid than the step that limits chemistry. Binding of Mg(2+) and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in alpha-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within alpha-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of alpha-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol beta deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors.     

Solution structure of the lyase domain of human DNA polymerase lambda

DNA polymerase lambda (pol lambda) is a recently discovered nuclear enzyme belonging to the pol X family of DNA polymerases that exhibits a 32% sequence identity with the nuclear DNA repair protein, pol beta. Structural modeling suggests that pol lambda contains the palm, fingers, thumb, and 8 kDa lyase domains present in pol beta, as well as an additional N-terminal BRCT domain and a serine-proline-rich linker that are presumably involved in protein-protein interactions. The 8 kDa domain of pol beta is important for DNA binding and contains the dRP lyase activity, which is the rate-limiting step in the single-nucleotide base excision repair (BER) pathway of damaged DNA. Recently, it was shown that the 8 kDa domain of pol lambda also contains the dRP lyase activity. To gain further insight into the catalytic mechanism of dRP removal by pol lambda, we have determined the solution structure of the 8 kDa lyase domain of human DNA pol lambda via multidimensional NMR methods and the ARIA program. The resulting structures exhibited a high degree of similarity with the 8 kDa lyase domain of pol beta. Specifically, the side chains of residues W274, R275, Y279, K307, R308, and K312 are in similar positions to the functionally important side chains of residues H34, K35, Y39, K60, K68, and K72 in the 8 kDa lyase domain of pol beta. This suggests that, on the basis of the proposed roles of these residues in pol beta, the corresponding pol lambda side chains may be involved in DNA binding and dRP lyase activity. The structural alignment of W274 (pol lambda) with H34 (pol beta) indicates that the former is probably involved in a similar base stacking interaction with template DNA at the position of the gap, in contrast with several previous proposals which aligned D272 with H34. In a few cases for which there is a nonconservative substitution in the sequence alignment, a structural comparison shows a positionally and, hence, probably a functionally equivalent residue, e.g., K60 in pol beta and K307 in pol lambda. Additionally, on the basis of the structural alignment obtained, several previously proposed mechanistic hypotheses can be evaluated.     

Localization of the deoxyribose phosphate lyase active site in human DNA polymerase iota by controlled proteolysis

Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.     

Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping

DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.     

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries,intrinsic affinities,and cooperativities

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.     

Kinetic mechanisms of rat polymerase beta-ssDNA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (Pol beta)(16) and (Pol beta)(5) ssDNA binding mode.

Kinetics of rat polymerase beta (pol beta) binding to the single-stranded DNA (ssDNA) in the (pol beta)(16) and (pol beta)(5) binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol beta)(16) binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol beta, and with a 10-mer which binds the enzyme exclusively in the (pol beta)(5) binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol beta)(16) binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: [formula-see text] A similar mechanism is observed in the formation of the (pol beta)(5) binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol beta)(16) binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol beta, prior to ssDNA binding.     

The primary DNA-binding subsite of the rat pol β. Energetics of interactions of the 8-kDa domain of the enzyme with the ssDNA

Interactions of the 8-kDa domain of the rat pol β and the intact enzyme with the ssDNA have been studied, using the quantitative fluorescence titration technique. The 8-kDa domain induces large topological changes in the bound DNA structure and engages much larger fragments of the DNA than when embedded in the intact enzyme. The DNA affinity of the domain is predominantly driven by entropy changes, dominated by the water release from the protein. The thermodynamic characteristics dramatically change when the domain is embedded in the intact polymerase, indicating the presence of significant communication between the 8-kDa domain and the catalytic 31-kDa domain. The diminished water release from the 31-kDa domain strongly contributes to its dramatically lower DNA affinity, as compared to the 8-kDa domain. Unlike the 8-kDa domain, the DNA binding of the intact pol β is driven by entropy changes, originating from the structural changes of the formed complexes.     

Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase alpha and beta and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure-function relationship of the synthetic sulfo-glycolipids to DNA polymerase alpha and beta and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase alpha with IC(50) values of 3-5 microM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.     

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.     

Identification and properties of the catalytic domain of mammalian DNA polymerase beta

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.     

Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.     

Recognition of template-primer and gapped DNA substrates by the human DNA polymerase beta

Interactions between human DNA polymerase beta and the template-primer, as well as gapped DNA substrates, have been studied using quantitative fluorescence titration and analytical ultracentrifugation techniques. In solution, human pol beta binds template-primer DNA substrates with a stoichiometry much higher than predicted on the basis of the crystallographic structure of the polymerase-DNA complex. The obtained stoichiometries can be understood in the context of the polymerase affinity for the dsDNA and the two ssDNA binding modes, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotide residues occluded by the protein in the complex. The analysis of polymerase binding to different template-primer substrates has been performed using the statistical thermodynamic model which accounts for the existence of different ssDNA binding modes and has allowed us to extract intrinsic spectroscopic and binding parameters. The data reveal that the small 8 kDa domain of the enzyme can engage the dsDNA in interactions, downstream from the primer, in both (pol beta)(16) and (pol beta)(5) binding modes. The affinity, as well as the stoichiometry of human pol beta binding to the gapped DNAs is not affected by the decreasing size of the ssDNA gap, indicating that the enzyme recognizes the ssDNA gaps of different sizes with very similar efficiency. On the basis of the obtained results we propose a plausible model for the gapped DNA recognition by human pol beta. The enzyme binds the ss/dsDNA junction of the gap, using its 31 kDa domain, with slight preference over the dsDNA. Binding only to the junction, but not to the dsDNA, induces an allosteric conformational transition of the enzyme and the entire enzyme-DNA complex which results in binding of the 8 kDa domain with the dsDNA. This, in turn, leads to the significant amplification of the enzyme affinity for the gap over the surrounding dsDNA, independent of the gap size. The presence of the 5'-terminal phosphate, downstream from the primer, has little effect on the affinity, but profoundly affects the ssDNA conformation in the complex. The significance of these results for the mechanistic model of the functioning of human pol beta is discussed.