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Thymidine kinase was isolated and purified 1600-fold from sea urchin (Strongylocentrotus intermedius) oocytes. The molecular mass of the enzyme is 66 kDa, pI is 5.2. The enzyme activity needs Mg2+, ATP and the corresponding phosphate acceptor. The pH optimum of the enzyme activity is at 9.0-9.5. The isolated enzyme does not exhibit any strict substrate specificity and can phosphorylate thymidine, deoxycytidine and some other pyrimidine nucleosides and their derivatives, the phosphorylation rate being maximal for thymidine, ATP or dATP. The TMP formed via the enzymatic reaction does not influence the thymidine kinase activity.

It was shown that 3'-deoxy-3'-aminothymidine and 2',3'-dideoxy-3'-aminoadenosine are effective inhibitors of DNA synthesis of sea urchin (Paracentrotus lividus) embryos during the early steps of development. The most probable mechanism of inhibition includes the blocking of the template-directed polycondensation, in which the inhibitors act as analogs of the substrates--thymidine and 2'-deoxyadenosine 5'-triphosphates. An efficient degradation of the newly synthesized DNA was observed during the inhibition.

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.

Ca2+-Mg2+-dependent deoxyribonuclease (deoxyribonucleate-5'-oligonucleotidehydrolase E. C. 3.1.4.5). Molecular weight of the enzyme is found to be 40 000 daltons isoelectric point--4.4. The enzyme degraded DNA only in the presence of bivalent cations. It hydrolyses preferentially native DNA with pH optimum 7.0-7.2 in the presence of Mg2+ ions. Ca2+ ions shift the pH optimum to 8.0-8.5. Combined addition of Ca2+ and Mg2+ ions results in a sinergic effect and changes the enzyme specificity to the secondary DNA structure. The enzyme hydrolyses both native and denatured DNA by the endonucleolytic type to form oligonucleotides with 5' terminal phosphate the content of tetra-octanucleotides being 80-85%.

DNA-polymerase beta was isolated from embryonic cells of the sea urchin S. intermedius and purified 1040-fold. The molecular weight of the enzyme is 40 000, sedimentation coefficient 3.2S, pI 8.5. The SH-reagent--N-ethylmaleimide--has no appreciable influence on the enzyme activity. The enzyme is thermolabile and needs four deoxyribonucleoside triphosphates, bivalent metal ions (Mg2+ or Mn2+) and primer template for its activity. The maximal activity is observed when a synthetic polymer--poly(dA).oligo(dT) is used. DNA-polymerase performs DNA synthesis via a distributive mechanism. In terms of physico-chemical properties, the enzyme can be related to DNA-polymerases beta.

An ATP-dependent DNAse was isolated from the cells of the sea urchin Strongylocentrotus intermedius embryos by chromatography on DEAE-cellulose and gel chromatography on Sepharose 4B. The enzyme was found homogeneous during polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined by gel filtration through Sepharose 6B and was equal to approximately 450,000. The sedimentation coefficient as determined by ultracentrifugation was equal to approximately 15S. The pH optimum during native DNA hydrolysis lies within the pH range of 6,5--9,0 and that during hydrolysis of denaturated DNA--within the pH range of 9,0--9,5. The enzyme was activated by ATP and dATP at the optimal concentration of 10(-4) M. Other nucleoside triphosphates did not substitute for ATP in this reaction. The hydrolysis of denaturated DNA occurred via the exonuclease way with a formation of short (di-, tri,- tetra- and penta-) oligonucleotides. The enzyme hydrolyzed native DNA according to the endonuclease type with predominant formation of high molecular weight polynucleotides.

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.

Ca2+, Mg2+-dependent DNAse has been isolated in a homogeneous state from the embryos of the sea urchin Strongylocentrotus intermedius. The enzyme hydrolyzes DNA only in the presence of bivalent metal ions and is inhibited by EDTA and EGTA. The most effective activators are Mn2+ and Co2+. In the presence of Mg2+ + EGTA, Ca2+, Ba+2 and Sr2+ the DNA is hydrolyzed in a lesser degree. Mg2+ + Ca2+ as well as Mg2+ + Sr2+ produce a synergic activating effect. The concentration of Ca2+ making up to one half of the maximal activity in the presence of 5 mM MgCl2 decreases with an increase in DNA concentration from 1.10(-4) M Ca2+ for 1,7 . 10(-5) M DNA-P up to 3,0 . 10(-5) M Ca2+ for 16,0. 10(-5) M DNA-P. The value of V for enzymatic hydrolysis of DNA does not depend on the concentration of Ca2+, while Km(app) for the enzyme increases from 2,0 . 10(-5) M DNA-P for 5 . 10(-4) M Ca2+ up to 25 . 10(-5) M DNA-P for 1 . 10(-5) M Ca2+. The Km(app) of the enzyme for Mg2+ + EGTA is 3.2 . 10(-5) M DNA-P, for Ca2+--0,77 . 10(-5) DNA-P.

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.