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 investi gation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, a nd when interpreting the data this does not allow taking into account the r elative contribution of some specific and nonspecific interactions to the t otal affinity of nucleic acids (NA) to enzymes. This often results in unjus tified overestimation of the role of specific enzyme-NA contacts in affinit y and specificity of enzyme action. In recent years we have developed new a pproaches to analysis of the mechanisms of protein-nucleic acid interaction s allowing quantitative estimation of the relative contribution of virtuall y every nucleotide unit (including individual structural elements) to the t otal affinity of enzymes to long DNA and RNA molecules. It is shown that th e interaction between enzymes and NA on the molecular level can be successf ully analyzed by the methods of synthesis and analysis, that is, step-by-st ep simplification or complication of the structure of a long NA-ligand. Thi s approach allows the demonstration that complex formation including format ion of contacts between enzymes and specific NA units can provide neither h igh affinity of the enzymes to NA nor the specificity of their action. Usin g a number of sequence-independent replication and repair enzymes specifica lly recognizing a modified unit in DNA and also some sequence-dependent top oisomerization and restriction enzymes as examples, it was shown that virtu ally all nucleotide units within the DNA binding cleft interact with the en zyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is p rovided by many weak additive interactions between these enzymes and variou s structural elements of the individual NA nucleotide units. At the same ti me, the relative contribution of specific interactions to the total affinit y of NA is rather small and does not exceed 1-2 orders of magnitude. Specif icity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: k(cat) i ncreases by 3-7 orders of magnitude when changing from nonspecific to speci fic NA. In the present work we summarized our experience in studies of enzy mes 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 poss ibilities for the study of protein-nucleic acid interactions on the molecul ar level.