NUCLEASES — group of the enzymes which are specifically attacking (a way of influence) nucleic acids and catalyzing splitting of internucleotide bonds in poly-or oligonucleotides without formation of inorganic phosphate; on the nature of catalytic action are phosphodiesterases. N are found in all live organisms. They possess an essential role in cellular metabolism and in a metabolism of an organism in general; they participate in digestion, various processes of exchange nucleic acids (see), a genetic recombination, correction of genetic damages (reparation) and protection of a cell from alien nucleinic to - t. The size of activity of N. in various fabrics and biol, liquids of the person matters for diagnosis of a number of diseases. So, many times over activities of a deoxyribonuclease (DNA-ase I) in blood serum observe increase at acute inflammatory processes in a pancreas, in particular at acute pancreatitis and especially its hemorrhagic form. Increase in activity of ribonuclease (RNA elements) in blood serum is noted at uraemia. Disturbance of activity of N. participating in processes of a reparation, presumably, plays the main role in a pathogeny of various forms of a serious hereditary illness — a pigmental xeroderma.
N find broad application in a wedge, practice. DNA-ase is used, e.g., for treatment of the nek-ry heavy viral infections caused by hl. obr. adenoviruses and viruses of herpes, nek-ry diseases of a nervous system, suppurative processes, meningitis (see. Deoxyribonucleases ). Drugs RNA elements were effective at treatment of a tick-borne encephalitis (see. Ribonucleases ).
Specific N. are often applied in biochemical, and molecular biol. researches of structure and functions nucleinic to - t and all genetic device of a cell.
N are found in many tissues and liquids of a body of the person: liver, spleen, thymus gland, saliva, blood serum. The growing young fabrics differ in high activity of N.
Though all N. on the nature of the action are phosphodiesterases, however the mechanism of a rupture of phosphodiester communication can be various. Therefore the group H. includes representatives of several classes and subclasses enzymes (see). First of all it is hydrolases of phosphodiesters (KF 3.1.4) — the enzymes catalyzing a rupture of internucleotide bonds by direct hydrolysis. Here all DNA-ases depolymerizing the molecules DNA, nucleases which are not showing noticeable specificity to a carbohydrate component nucleinic to - you and attacking as DNA belong. and RNA, and also the nek-ry RNA elements catalyzing a depolymerization of molecules RNA. Other (big) part of RNA az carries out splitting of molecules RNA not by direct hydrolysis, and through the intramolecular perefosfori-lirovaniye preceding it.
On a way of the attack of substrate H. divide into two categories: endonukleolitichesky (endonucleases) and ekzonukleolitichesky (ekzonuklea-za); these terms are offered in 1959 by Lyaskovsky (M. to Laskowski). Endonucleases split a polynucleotide between internal links of a polymer chain. At the same time a certain degree of selectivity of enzyme — specificity to the chemical nature of the bases which are in the neighbourhood with the attacked bonds is often shown. Can be examples of strictly specific endonucleases pyrimidinspecific RNK-aza I (KF 18.104.22.168) of animal fabrics and guaninspetsifichny RNK-aza TI from Aspergillus oryzae (a guanilribonukleaza; KF 22.214.171.124). DNA-ases, as a rule, have no strict specificity concerning the nitrogen bases surrounding the attacked communication in molecule DNA only distinctions in speeds of hydrolysis depending on type of the basis are observed. On the mechanism of endonukleolitichesky splitting of DNA endonucleases divide into the enzymes making a rupture of two complementary chains in the same place (the two-shock mechanism) and in a random way in different sites of each of chains so gaps in all double-helix molecule are formed not at once but only on condition of symmetrically located gaps in complementary chains (the one-shock mechanism).
Ekzonukleaza catalyze consecutive eliminating of mononucleotides from one of the ends of a polynucleotide chain. The depolymerization of a molecule nucleinic to - you results up to oligo-or mononucleotides. The majority ekzonukleaz has no strongly expressed specificity in relation to nitrogen bases of the nucleotides next to the attacked bonds. Nek-ry ekzonukleaza are capable to hydrolyze both types nucleinic to - t though affinity to DNA and RNA at them usually unequal. The direction of movement of an ekzonukleaza along a chain of a polynucleotide (3' — >5' or 5' — >3') variously for different enzymes.
N.'s division on endo-and ekzonukleaza sufficiently conditionally. Detailed researches of high cleaning drugs H. indicate that actually individual enzyme can have ability to catalyze as endo-, and ekzonukleolitiche-sky splitting depending on structure of macromolecular substrate (such action the nuclease of micrococci, KF 126.96.36.199 allocated from pathogenic strains of Staphylococcus aureus possesses, e.g.).
Besides, changes in the nature of the enzymatic attack (endo-or ekzo-) and in specificity to the nitrogen bases adjoining on the attacked communication at early and final stages of reaction are possible.
As essential criterion for N.'s classification serves the provision of carbon atom in the rest of a ribose or desoxyribose of a nucleotide (3' - or 5 '-), at to-rogo there is a splitting of phosphodiester communication. Depending on it hydrolysates with trailer 5 '-or Z' - monophosphate respectively are formed. N are characterized by also various sensitivity to secondary structure of substrate.
In special category H. it is necessary to emit so-called enzymes of restriction, or restriction endonucleases (restriktaza) found so far only in microorganisms, but existence to-rykh has probably basic all-biological value. So far them it is described apprx. 150; by 1978 more than 50 restriction endonucleases are recognized as individual enzymes. These enzymes, having exclusive specificity, «learn» the nucleotide sequences of strictly certain structure and «cut» a molecule of double-helix DNA on large fragments. As a result a set of the fragments inherent in this restriktaza characteristic by the size and structure is always formed of this DNA. Many restriktaza split chains of double-helix DNA in incoincident points — with shift in several nucleotides.
N.'s most represents small globular proteins with the expressed main properties [the exception is made by large ATP-dependent DNA-ases, their pier. the weight (weight) reaches 350 000].
DNA-ases and nonspecific endonucleases usually of a termolabilna, have optikhmum pH in neutral or alkaline area, are activated by bivalent cations and inhibited by kompleksobrazuyushchy (chelating) agents. RNA elements are in most cases active at acid and alkaline pH values; are activated one - and bivalent cations. Nek-ry RNA elements do not demand activators and differ in high heat stability (e.g., RNK-aza from a pancreas of the person shows the maximum activity at 65 ° and maintains without noticeable change of activity short-term heating to 100 °).
N.'s localization is connected with fiziol. functions of these enzymes. The N of microorganisms located about a cell wall and cosecreted to the environment participate in food of bacteria, split the substrates which are not getting inside cells. Extracellular DNA-ases of pathogenic bacteriums, perhaps, play a role in a pathogeny of nek-ry infections. For the pathogenic microorganisms emitting exotoxins correlation between toxin production and secretion of DNA-ase is established. Such endonucleases of animals as RNK-aza I and DNA-ase of I of a pancreas, glands which are formed in zimogenny granules and cosecreted in pancreatic juice provide splitting nucleinic to - t at digestion and assimilation of their components with a cell. These enzymes participate also in destruction of fabrics and microorganisms at various patol, processes. Intracellular N. can be a part of the fermental systems protecting a cell from an infection alien DNA, e.g. DNA of viruses. In kernels, cytosol, mitochondrions and lysosomes N. which are performing regulatory functions or directly participating in processes of synthesis and disintegration, maturing (processing), a reparation and recombination of genetic material are localized. The acid N. which are contained in lysosomes work after escaping them through a lysosomic membrane at nek-ry fiziol, conditions of a cell and at its destruction.
Nek-ry DNA polymerases have ekzonukleazny activity, thanking a cut with their participation the inoculating end of DNA of not coupled or incorrectly coupled nucleotides is removed that provides correctness of copying of a DNA matrix in the course of replication.
In 1966 W. L. Carrier and Setlou (R. Century of Setlow) showed that damages to molecule DNA, voznrshayushchy at radiation of bacteria UV rays and expressed in education a dimeasure of thymine, can be corrected by cells by «cutting» dimer from molecule DNA. The catalytic mechanism of this process is provided N. Izvestna three systems correcting or compensating the damages of DNA caused by ionizing radiation, chemical agents etc. It is the Dark reparation, a reparation as a result of exchange of fragments of DNA at a recombination, photoreactivation — splitting of dimer of thymine the enzymes activated by light with wavelength apprx. 400 nanometers (see. Reparation of genetic damages ).
At the first stages of a tempo reparation specific endonuclease «cuts» a defective chain of two-siiralnoy DNA near the place of damage. The single gap which is formed in a chain uses an ekzonukleaz, «cutting out» the area containing the damaged site. After «cutting» or along with this process there is reparative a synthesis of DNA which is carried out by a DNA polymerase, edges uses as a matrix the unimpaired chain, and, at last, sewing together by enzyme ligase of the ends of the old and again synthesized chains.
Part of damages of a chain of DNA caused by alkylating agents or ionizing radiation cannot be recovered on the mechanism of a dark reparation and improves as a result of exchange of the damaged site of DNA on unimpaired in the course of a recombination of genes (see. Recombination ). The increasing value is gained by ideas of a recombination as about the process connected with «recognition» of certain sites of DNA specific N.
Bolshinstvo of methods of definition of activity of N. is based on sedimentation of not degraded DNA or RNA from reaction mixture with the subsequent determination of amount of acidsoluble reaction products — oligo-and mononucleotides or on measurement of hyperchromic effect (increase of the optical absorption) at 260 nanometers caused by a depolymerization nucleinic to - you. In a wedge, practice activity of DNA-ase is often determined by change viscosity (see) solutions of polymeric DNA in the course of its enzymatic degradation. As the first scission step ribonucleic to - t the RNA Hadean (formation of cyclophosphates) goes with a much bigger speed, than the second stage (actually hydrolysis), definition the RNA Hadean of activity in nek-ry cases is carried out, measuring the hypochrom-ism (reduction of optical absorption) at 300 nanometers of RNA solution caused by formation of cyclic phosphates.
Bibliography: Davidson J. Biochemistry of nucleic acids, the lane with English, page 193, M., 1976; Nucleases of microorganisms, under the editorship of A. M. Bezborodov, M., 1974; Sh and p about V. S t. Nucleases, M., 1968, bibliogr.; The enzymes, ed. by P. D. Boyer, v. 4, p. 153, N. Y. — L., 1972.
P. L. Ivanov.