REVIEW: 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

D. V. Bugreev and G. A. Nevinsky^*

Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, pr. Akademika Lavrent´eva 8, Novosibirsk, 630090 Russia; E-mail: nevinsky@niboch.nsc.ru

^* To whom correspondence should be addressed.

Received May 15, 1998; Revision received November 28, 1998

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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: k_cat 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.

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KEY WORDS: mechanism of protein--nucleic acid interactions, method of step-by-step complication of the structure of nucleic acid, possibilities of the method in investigation of enzymes

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