The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Interaction of endonuclease ecoRI with short specific and nonspecific oligonucleotides

The interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-step simplification in their complexity. Orthophosphate (KI = 31 mM), 2-deoxyribose 5-phosphate (KI = 4.6 mM) and different dNMPs (KI = 2.1-2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN)n (n = 1-6) by one nucleotide unit resulted in the increase of their affinity by a factor of approximately 2.0. Weak nonspecific electrostatic contacts of EcoRI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between EcoRI and d(pN)n is no more than 2 orders of magnitude of a total ODN's affinity.     

The mechanism of the supercoiled DNA recognition by eukaryotic type I topoisomerases. I. The enzyme interaction with nonspecific oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.     

The Mechanism of the Supercoiled DNA Recognition by the Eukaryotic Type I Topoisomerases: I. The Enzyme Interaction with Nonspecific Oligonucleotides

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.     

Interaction of the human topoisomerase I-DNA complex with oligo-1,3-thiazolecarboxamides and their oligonucleotide conjugates

Nonnatural thiazole-containing oligopeptides (TCOs) bind to the DNA minor groove and inhibit the reaction catalyzed by human topoisomerase I (TopoI). The effect is directly proportional to the number of thiazole monomers in TCO. Several TCOs with three or four thiazole monomers act 3–10 times more efficiently than distamycin A, a natural antibiotic containing pyrrole rings. Additional groups at the N and C termini only slightly affect TopoI inhibition by TCO. The inhibitory effect of TCOs is higher than that of homo-or heterooligopeptides containing imidazole or pyrrole monomers, and the most potent are oligopeptide-oligonucleotide conjugates. The plausible causes of the different effects of distamycin and the nonnatural peptides on DNA relaxation catalyzed by TopoI are discussed.     

Possibilities of the method of step-by-step complication of ligand structure in studies of protein--nucleic acid interactions: mechanisms of functioning of some replication, repair, topoisomerization, and restriction enzymes

X-Ray structure analysis is one of the most informative methods for investigation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, and when interpreting the data this does not allow taking into account the relative contribution of some specific and nonspecific interactions to the total affinity of nucleic acids (NA) to enzymes. This often results in unjustified overestimation of the role of specific enzyme--NA contacts in affinity and specificity of enzyme action. In recent years we have developed new approaches to analysis of the mechanisms of protein--nucleic acid interactions allowing quantitative estimation of the relative contribution of virtually every nucleotide unit (including individual structural elements) to the total affinity of enzymes to long DNA and RNA molecules. It is shown that the interaction between enzymes and NA on the molecular level can be successfully analyzed by the methods of synthesis and analysis, that is, step-by-step simplification or complication of the structure of a long NA-ligand. This approach allows the demonstration that complex formation including formation of contacts between enzymes and specific NA units can provide neither high affinity of the enzymes to NA nor the specificity of their action. Using a number of sequence-independent replication and repair enzymes specifically recognizing a modified unit in DNA and also some sequence-dependent topoisomerization and restriction enzymes as examples, it was shown that virtually all nucleotide units within the DNA binding cleft interact with the enzyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is provided by many weak additive interactions between these enzymes and various structural elements of the individual NA nucleotide units. At the same time, the relative contribution of specific interactions to the total affinity of NA is rather small and does not exceed 1-2 orders of magnitude. Specificity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: kcat increases by 3-7 orders of magnitude when changing from nonspecific to specific NA. In the present work we summarized our experience in studies of enzymes by the method of step-by-step complication of the ligand structure and performed a detailed analysis of the features of this approach and its possibilities for the study of protein--nucleic acid interactions on the molecular level.