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.

Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of human DNA topoisomerase I (topo I) with specific sequence oligodeoxynucleotides (ODNs) of different length and structure has been investigated. All the ODNs used were shown to be effective enzyme inhibitors and to inhibit the topo I catalyzed relaxation of scDNA in a competitive manner. Among two DNA regions (A and B) required for topo I-mediated DNA cleavage, the former was found to display the higher affinity for the enzyme. The enzyme's affinity for ODNs corresponding to the scissile strand (five and nine nucleotide units in length) is about 2-4 orders of magnitude higher than that for non-specific ODNs of the same length. Topo I can efficiently recognize even extremely short specific ODNs containing only two or three bases (AGA and pAG, Ki = 15 and 60 microM, respectively): the sequence AAGA (Ki = 10 microM) is essential for tight DNA binding to topo I. The affinities of ODNs corresponding to the non-scissile strand are significantly lower. The ligand's affinity increases with its length. Additionally, about a ten-fold enhancement of specific sequence affinity occurs due to stable duplex formation during enzyme preincubation with ligands before addition of scDNA. We believe the possibility of using the short specific oligonucleotides and its derivatives as topoisomerase I-targeting drugs could not be excluded.

The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase alpha.

DNA synthesis with various deoxyribo homo- and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)n, primers (n = 1-10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template-primer structure in solution was analyzed and the objective laws of the changes of the KM and Vmax values were revealed.

Recognition of the primers containing different modified nucleotide units by the Klenow fragment of DNA polymerase I from E coli.

A comparison of Km values and maximal rates of extension (Vmax) for primers containing different modified bases or mismatches, and fully complementary primers of the same length catalyzed by the Klenow fragment of E coli DNA polymerase I was carried out. Base modifications include T-T dimers and apurinic sites. In the case of mismatch, the number of complementary bases from the 3'-terminus to the non-complementary nucleotide determines the efficiency of substrate incorporation, which is a measure of degree of interaction of the enzyme with its primer template. Differently, removal of one base in any position from the 3'-terminus of the primer is equivalent to shortening of the primer by one nucleotide unit, and decreases the affinity to the enzyme by 1.8-fold. Since apurinic sites fail to interfere with the efficiency of DNA synthesis, we suppose that the Klenow fragment of E coli DNA polymerase I does not participate in the correction of DNAs containing apurinic nucleotides units. Finally, the efficiency of elongation of the d(p primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10m primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

[Isolation of DNA polymerase beta from human placenta and study of its substrate specificity]. / Vydelenie DNK-polimerazy beta iz platsenty cheloveka i issledovanie ee substratnoi spetsifichnosti.

A method of purification of DNA polymerase beta with a specific activity of 1300 units/mg from human placenta was developed. The enzyme preparations do not contain any other DNA polymerase activities and any nuclease contaminations degrading nucleic acids. On the basis of analysis of several standard parameters we conclude that the purified enzyme is polymerase beta. The optimal conditions of polymerization were established, and a comparison of the relative rates of polymerization with various template-primer complexes was carried out. Activated DNA was shown to be the optimal substrate in the presence of MgCl2, and poly(dA).oligo(dT) in the presence of MnCl2. The activation energies of polymerization for different template-primers were estimated.

[Recognition of primers, containing noncomplementary nucleotides and units, lacking bases, by the Klenow fragment of Escherichia coli DNA polymerase I]. / Uznavanie fragmentom klenova DNK-polimerazy I iz Escherichia coli praimerov, soderzhashchikh nekomplementarnye matritse nukleotidy i aven'ia, lishennye osnovanii.

A comparison was carried out for the Km and maximal rates of conversion on (Vmax) with primers containing noncomplementary bases, as well as primers without several bases, and fully complementary primers of the same length. The number of complementary bases from the 3' end to the noncomplementary nucleotide was shown to determine the efficiency of interaction (and conversion) of the primers containing noncomplementary bases with enzyme. The DNA polymerase practically does not discriminate between the primers without one or two bases and the fully complementary primers. Elimination of one base in any position from the 3' end of the primer is equivalent to shortening of the primer by one nucleotide unit, and leads to a decrease in the affinity by a factor of 1.8. We suppose that DNA polymerase does not participate in primer mistake correction during the repair process if the DNA contains apurinic or apyrimidinic nucleotide units.

[Recognition of T-T cyclobutane pyrimidine dimers in the form of primers by DNA polymerases]. / Uznavanie DNK-polimerazami T-T tsiklobutanovykh pirimidinovykh dimerov v sostave praimerov.

A study was made of the efficiency of primer conversion catalyzed by human placenta DNA polymerase on the length of primer. The dependence of -log Kd and Vmax on the number of mononucleotide units (n) of primer were shown to be linear up to n <--> 10. Each mononucleotide unit of the primer enhanced its affinity by a factor of 2.3 and the maximal polymerization rate by a factor of 1.3. For the first time estimation was made for the contribution of replication enzymes: human placenta DNA polymerase and E. coli DNA polymerase I (as well as AMV reverse transcriptase) to a decrease in the amount of T-T dimers in DNA emerging after UV- or gamma-irradiation. The efficiency of elongation of the d(pT)10 primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10 primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase.

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

The affinity of the Klenow fragment of E. coli DNA-polymerase 1 to primers containing bases noncomplementary to the template and hairpin-like elements.

The Km and Vmax values for a set of primers: d(pT)n (pC) (pT)m (n = 3-9, m = 0-7) and d(pT)4 (pCpG)k (pT)4 (k = 1-5) have been estimated. Poly(dA) was used as a template. The number of complementary bases from the 3' end to a noncomplementary ones was shown to determine the efficiency of interaction of d(pT)n (pC) (pT)m with the Klenow fragment. Oligonucleotides d(pT)4 (pCpG)k (pT)4, in solution forming duplexes containing hairpin-like elements, show a higher affinity to the enzyme than control d(pT)4, d(pT)8 and d(pT)n (pC) (pT)m primers. For example, the Km value (1.1 nM) for d(pT)4 (pCpG)5 (pT)4 is about 14,000 and 200 times lower than those for d(pT)4 and d(pT)8, respectively. Possible reasons for such an abnormally high affinity of the above primers are discussed.

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.

Pt(2+)-containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase alpha from human placenta. The values of Kd and Gibb's energy (delta G degree) for the complexes of oligodeoxyribonucleotides and their derivatives with the template sites of these enzymes were determined from the effects protecting the enzyme from inactivation by Pt(2+)-containing oligonucleotides. Kd and delta G degree values of the complexes made by DNA polymerases and orthophosphate, triethylphosphate, d(pC)n, d(pT)n, d(pG)n, d(pA)n (where n = 1-25), heterooligonucleotides of various length and structure, and oligothymidylates with partially and completely ethylated internucleotide phosphates were evaluated. The obtained data enabled us to suggest 19-20 mononucleotide units of the template to interact with the protein. Only one template internucleotide phosphate forms a Me(2+)-dependent electrostatic contact (delta G = -1.1...-1.7 kcal/mol) and a hydrogen bond (delta G = -4.4...-4.9 kcal/mol) with the enzyme. It is likely that the mononucleoside units of the template form hydrophobic contacts with the enzymes. The efficiency of such interaction changes with the hydrophobicity of the bases: C less than T less than G approximately A. For both homo- and heterooligonucleotides the contributions of nucleoside units to the affinity of the templates to the enzymes is due to the complementary interactions with the primers. A hypothetical model for the template-primer interaction with DNA polymerases is suggested.

DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition.

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.

[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. / Fragment klenova DNK-polimerazy I iz E. coli. III. Rol' mezhnukleotidnykh fosfatnykh grupp matritsy v ee sviazyvanii s fermentom.

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive. Labelling took place only when the combination of the reagent and [alpha-32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the sigma subunit was also labelled.

[Plasmid vector pSSC1 for cloning synthetic polynucleotides and their regeneration with a unique nucleotide sequence]. / Plazmidnyi vektor pSSC1 dlia klonirovaniia sinteticheskikh polinukleotidov i ikh regeneratsii s unikal'noi nukleotidnoi posledovatel'nost'iu.

For subcloning separate synthetic gene fragments, a plasmid vector pSSC1 was constructed by inserting a synthetic polylinker into plasmid pBR 327 at the EcoRI-PstI sites. There are two FokI and HgaI sites at the ends of this polylinker in the opposite orientation, with the EcoRI and PstI sites between them. DNA fragments cloned at the EcoRI and PstI sites can be regenerated by either FokI or HgaI, the EcoRI and PstI sites being deleted from the cloned sequences. Such fragments have unique cohesive ends that allows their directed ligation into longer DNA (genes).