The Mechanism of Recognition of Templates By DNA Polymerases From Pro- and Eukaryotes as Revealed By Affinity Modification Data

Abstract

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.

Interaction of Endonuclease Eco RI with Short Specific and Nonspecific Oligonucleotides

“…Two symmetrical DNA-binding "channels" of each EcoRI subunits are at 3.5 Ê A apart and two different strand of ds DNA bind to different enzyme subunits (21). Therefore, it seems most likely that the interaction of EcoRI subunits with DNA strands may weaken the complementary interactions between them, even for speci c DNA as was also shown for replication (1,9,12), repair enzymes (5-7), and topoisomerases (2; 13). Thus, as in the case of other enzymes, the complementary interactions of the strands outside the EcoRI molecule may be important for stabilization of the enzyme interactions with a fragment of DNA complexed with the enzyme (or complementary interaction between the strands), and such interactions can lead to an increase of the af nity of EcoRI for ds d(pN) n at n > 10.…”

“…4). Orthophosphate (P i ) was shown earlier to be the minimal ligand of special subsites of DNA-binding centers for the enzymes involved in replication (9,11,12) or repair (4-7 ), and DNA topoisomerase I (13). One of 6-7 subsites of EcoRI also has a high af nity to P i (I 50 D 31 mM).…”

“…Recently we have developed new methods which allow us to estimate relative ef ciency of interaction of a DNA-dependent enzyme with each individual nucleotide unit (and its structural elements) of any speci c and nonspeci c ss and ds DNA (for review see 1,2). A relative contribution of internucleotide phosphate groups, speci c and nonspeci c bases, single strands, and de nite sequences of ds DNAs to the enzyme's af nity was estimated for several repair (3)(4)(5)(6)(7), replication enzymes (8)(9)(10)(11)(12), and DNA topoisomerase I (13). All analyzed enzymes were shown to form weak, additive, nonspeci c contacts with internucleotide phosphate groups of all nucleotide units of ss and/or ds d(pN) n , which lie within their DNA binding clefts (depending on the enzyme, n D 10-20).…”

Interaction of primer tRNALys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

“…We showed earlier the important role of the 3'-end OH group )f the primer in its interaction with pro-and eucaryotic DNA ~olymerases [12,15,[22][23][24][25]. Here our aim was to estimate the :ontribution of the 3'-terminal CCA of tRNA in the interaction ~ith HIV-1 RT p66/p51.…”

Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNALys3 and Affinity Modification of The Enzyme by tRNALys3 Derivatives

“…Reverse transcriptase was strongly protected by tRNA from covalent attachment of [5'-32P]tRNA-R, in the presence or absence of poly(A). Protection of the enzyme by tRNA against covalent binding of [5'-'2P]tRNA-R, in the presence of poly(A) was used for evaluation of tRNA affinity to the reverse transcriptase according to Kolocheva et al (1991). In the presence of poly(A), tRNA interacts with the heterodimer showing different affinities (K,,, = 70 nM, Kd2 = 190 nM).…”