DNA-binding Subsite Research Articles

Interactions of the DNA Polymerase X From African Swine Fever Virus with the ssDNA. Properties of the Total DNA-Binding Site and the Strong DNA-Binding Subsite

Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. Direct binding experiments were performed with fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The primary DNA-binding subsite of the enzyme, which establishes the contact with the nucleic acid independent of the DNA conformation, is located on the C-terminal domain of the enzyme. Association of the bound DNA with the weak DNA-binding subsite on the catalytic N-terminal domain, finalizes the engagement of the total DNA-binding site of the enzyme in interactions with the nucleic acid. Moreover, it induces a large topological change in the structure of the bound ssDNA. The intrinsic free energy of binding of the total DNA-binding site is not a simple sum of contributions from the strong and the weak DNA-binding subsites. Large positive enthalpy changes accompanying the ASFV pol X - ssDNA association indicate that specific conformational changes of the complex are induced by the engagement of the weak DNA-binding subsite on the N-terminal domain. The unfavorable enthalpy changes are offset by large entropy changes accompanying the DNA binding to the strong DNA-binding subsite and the total DNA-binding site, and, predominantly, result from the release of the water molecules.

The Primary DNA-Binding Subsite of the Rat Pol Beta. 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.

The N-Terminal Domain of the Escherichia coli PriA Helicase Contains Both the DNA- and Nucleotide-Binding Sites. Energetics of Domain–DNA Interactions and Allosteric Effect of the Nucleotide Cofactors

Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein--double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer--single-stranded DNA (ssDNA) complex is ~13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3'-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain--N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

Interactions of the DNA polymerase X from African Swine Fever Virus with the ssDNA. Properties of the total DNA-binding site and the strong DNA-binding subsite

Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. The primary DNA-binding subsite of the enzyme, independent of the DNA conformation, is located on the C-terminal domain. Association of the bound DNA with the catalytic N-terminal domain finalizes the engagement of the total DNA-binding site of the enzyme and induces a large topological change in the structure of the bound ssDNA. The free energy of binding includes a conformational transition of the protein. Large positive enthalpy changes accompanying the ASFV pol X–ssDNA association indicate that conformational changes of the complex are induced by the engagement of the N-terminal domain. The enthalpy changes are offset by large entropy changes accompanying the DNA binding to the C-terminal domain and the total DNA-binding site, predominantly resulting from the release of water molecules.

The Escherichia Coli PriA Helicase Specifically Recognizes Gapped DNA Substrates

The primosome is a multi-protein-DNA complex that catalyzes the priming of the DNA during the replication process. In Escherichia coli, PriA helicase plays a fundamental role in the initiation of the ordered assembly of the primosome. PriA is involved in recombination and repair processes being a major factor that initiates the restart of the stalled replication fork at the damaged DNA sites. This happens, presumably, through the recognition of the damaged DNA site structure though the nature of this recognition process is unknown. Here we present quantitative studies of the ssDNA gap recognition by the PriA helicase and the effect of the nucleotide cofactors on the recognition process. The data indicate a surprisingly low minimum total site size of the enzyme in the gap complex, which is ∼7 nucleotides or bp as compared with the site size of ∼20 nucleotides of the enzyme-ssDNA complex. The low stoichiometry indicates that the helicase exclusively engages the strong DNA-binding subsite in the gap complex and assumes a very different orientation, compared with the complex with the ssDNA. The PriA helicase binds the ssDNA gaps with 4-5 nucleotides with the highest affinity without engaging in cooperative interactions with the enzyme molecules associated with the surrounding dsDNA. Binding of ADP to strong and weak nucleotide-binding sites of the enzyme profoundly affects the affinity and stoichiometry of the helicase-gapped DNA complex. These observations are of fundamental importance for understanding of the enzyme mechanisms in both replication and recombination processes.

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.

The Escherichia coli PriA Helicase–Double-Stranded DNA Complex: Location of the Strong DNA-Binding Subsite on the Helicase Domain of the Protein and the Affinity Control by the Two Nucleotide-Binding Sites of the Enzyme

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA–dsDNA complex is only 5 ± 1 bp, that is, dramatically lower than 20 ± 3 nucleotides occluded in the enzyme–single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA–dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.

The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates: EFFECT OF THE TWO NUCLEOTIDE-BINDING SITES OF THE ENZYME ON THE RECOGNITION PROCESS

Energetics and specificity of interactions between the Escherichia coli PriA helicase and the gapped DNAs have been studied, using the quantitative fluorescence titration and analytical ultracentrifugation methods. The gap complex has a surprisingly low minimum total site size, corresponding to approximately 7 nucleotides of the single-stranded DNA (ssDNA), as compared with the site size of approximately 20 nucleotides of the enzyme-ssDNA complex. The dramatic difference in stoichiometries indicates that the enzyme predominantly engages the strong DNA-binding subsite in interactions with the gap and assumes a very different orientation in the gap complex, as compared with the complex with the ssDNA. The helicase binds the ssDNA gaps with 4-5 nucleotides with the highest affinity, which is approximately 3 and approximately 2 orders of magnitude larger than the affinities for the ssDNA and double-stranded DNA, respectively. In the gap complex, the protein does not engage in cooperative interactions with the enzyme predominantly associated with the surrounding dsDNA. Binding of nucleoside triphosphate to the strong and weak nucleotide-binding sites of the helicase eliminates the selectivity of the enzyme for the size of the gap, whereas saturation of both sites with ADP leads to amplified affinity for the ssDNA gap containing 5 nucleotides and engagement of an additional protein area in interactions with the nucleic acid.

Mechanism of NTP Hydrolysis by the Escherichia coli Primary Replicative Helicase DnaB Protein. 2. Nucleotide and Nucleic Acid Specificities

The kinetic mechanism of NTP binding and hydrolysis by the Escherichia coli replicative helicase, the DnaB protein, in the absence and presence of the single-stranded DNA (ssDNA), has been quantitatively examined using the rapid quench-flow technique, under single-turnover conditions. In the case of both the free helicase and the enzyme-ssDNA complexes, the mechanism is independent of the type of base of the cofactor or the DNA; the bimolecular association is followed by the reversible chemical hydrolysis and subsequent conformational transition of the enzyme-product complex. The NTP hydrolysis step is significantly faster for the purine than for the pyrimidine cofactor, both in the absence and in the presence of the DNA. The temperature effect indicates that the nature of intermediates of the purine nucleotide, ATP, is different from the nature of the analogous intermediates of the pyrimidine nucleotide, CTP. Nevertheless, both types of cofactors seem to approach a similar "exit" state at the end of the reaction. The effect of ssDNA on the kinetics of NTP hydrolysis depends on the type of nucleotide cofactor and the base composition of the DNA and is centered at the hydrolysis step. Homoadenosine ssDNA oligomers are particularly effective in increasing the hydrolysis rate. The allosteric signal from the DNA, which activates the NTP hydrolysis, comes predominantly from the strong DNA-binding subsite. The role of the weak DNA-binding subsite is to modulate the allosteric effect of the strong subsite. The significance of these results for the mechanism of the free energy transduction by the DnaB helicase is discussed.

Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) base-pairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme–ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein–protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.

Dynamics of Gapped DNA Recognition by Human Polymerase β

Kinetics of human polymerase beta 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 approximately 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 PO(4)(-) 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.

Kinetic mechanisms of rat polymerase β-ssDNA interactions. quantitative fluorescence stopped-flow analysis of the formation of the (pol β)16 and (pol β)5 ssDNA binding mode

Kinetics of rat polymerase β (pol β) binding to the single-stranded DNA (ssDNA) in the (pol β)16 and (pol β)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 β)16 binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol β, and with a 10-mer which binds the enzyme exclusively in the (pol β)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 β)16 binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations:Rat polβ+ssDNA↔k−1k1(P16−ssDNA)1↔k−2k2(P16−ssDNA)2↔k−3k3(P16−ssDNA)3 A similar mechanism is observed in the formation of the (pol β)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 β)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 β, prior to ssDNA binding.