Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´′- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´′-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.     

Counterion dependent variation of DNA secondary structure in (A . T) clusters: evidence by use of netropsin as a structural probe.

The interaction of the oligopeptide antibiotic netropsin (Nt) with (A . T) regions of DNA is characterized by a spectrum of discrete modes. This has been revealed by viscometric analysis, at 20 degrees C and 0.2 M "counterions", for NaDNA in a preceding and for NH4DNA in this paper. The increase of DNA contour length as induced by one Nt molecule was found to depend on the special mode only, while the respective stiffening is generally higher for NH4DNA. The latter property is interpreted in terms of an enhanced flexibility, relative to that of NaDNA, of the (A . T) cluster segments before complex formation. For some of the interaction modes of the DNA-Nt systems a difference in the number of corresponding binding sites has been observed. This phenomenon is understood by assuming an influence of the counterion species upon existing equilibria between different forms of the (A . T) cluster secondary structure. Not less than 5 to 10% of the total DNA are effected in this manner. Upper limits for the local differences in the axial rise per base pair are 0.04 nm and 0.02 nm.     

Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference

Essential nucleotide contacts between the SV40 large T (tumor) antigen and binding sites I and II on the SV40 genome have been inferred from in vitro methylation- and ethylation-interference experiments. Each site contains two clusters of guanine residues that reduce the specific binding of T antigen when modified. Methylation at any one of nine guanines within site I or any one of five guanines within site II severely interferes with the interaction of T antigen with each respective site. Methylation at any one of a second group of five guanines within site II results in an appreciably weaker effect on the binding of T antigen. A similar inhibitory effect on binding is observed upon ethylation of adjacent phosphate residues. Although there are significant differences in the nucleotide sequence of the two binding sites, the pattern of protein contacts is strikingly similar between sites I and II. Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.     

Multimode Interaction of Hoechst 33258 with Eukaryotic DNA; Quantitative Analysis of the DNA Conformational Changes

Abstract

The interaction of the minor groove binding ligand Hoechst 33258 (Hoe) with natural DNA was investigated by high resolution titration rotational viscometry. Analysis of the concomitant DNA conformational changes was performed with two DNA samples of sufficiently different molar mass M, at 4°C, 22°C and 40°C, for Hoe/DNA-P ratios below r = 0.02. In this narrow r range several interaction modes could be resolved. The measured conformational changes were quantified in terms of relative changes of both apparent DNA persistence length, Δa/a, and hydrodynamically operative DNA contour length, ΔL/L. Δa/a(r) primarily is a measure of ligand-induced DNA helix stiffening, but both, Δa/a(r) and ΔL/L(r), generally depend also on ligand binding induced DNA bending or DNA unbending. The essential difference obviously is that Δa/a(r) is influenced by the randomly distributed helix bends and ΔL/L(r) by phased ones. The measurements performed at different temperatures deliver informations about existence and temperature dependent abolition of intrinsic helix curvature.

Both Hoe and netropsin (Nt) prefer binding to AT rich DNA segments, which are candidates for intrinsic DNA helix bends. But our data for Hoe interaction with calf thymus DNA (ctDNA) show characteristic differences to those for Nt-ctDNA interaction. Especially for Hoe, the mode of highest affinity is saturated already at a ligand concentration of roughly 1 nM (r = 0.0015 Hoe/DNA-P). It exhibits an unusually strong temperature dependence of the conformational DNA response. A Hoe-Nt competition experiment shows that Hoe binding to the sites of the very first Hoe mode is almost unaffected by bound Nt. But Hoe binding to the sites of the following Hoe modes does not occur due to the competition with Nt. Thus this mode of strongest Hoe-DNA interaction reflects a unique mechanism, possibly of high relevance for gene regulatory systems.     

The effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

Nt.BspD6I nicking endonuclease stimulates template/primer-independent DNA synthesis by Bst DNA polymerase. Template/primer-independent DNA synthesis may be one of the reasons for the formation of nonspecific products in certain DNA amplification reactions, especially those involving nicking endonucleases. Expansion of the range of DNA amplification procedures performed in the presence of nicking endonucleases makes the search for template/primer-independent DNA synthesis inhibitors highly relevant. The present work has shown that a single-strand DNA binding protein from E. coli does not affect template/primer-independent DNA synthesis regardless of the presence or absence of Nt.BspD6I. A single-stranded DNA-binding protein coded by gene 32 from bacteriophage T4 completely inhibits template/primer-independent DNA synthesis in the absence of nicking endonuclease. If nicking endonuclease is present, the protein does not suppress the synthesis of the specific product but causes a significant decrease of the amount of template/primer-independent DNA synthesis products.     

Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases.

The complete amino acid (aa) alignment of the N-terminal domain of 33 DNA-dependent DNA polymerases encompassing the putative segments Exo I, Exo II and Exo III, proposed by Bernad et al. [Cell 59 (1989) 219-228] to form a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases, allowed us to identify and/or correct some of the most conserved segments (Exo I, II and III) in certain DNA polymerases. In particular, the aa region of T4 DNA polymerase and other eukaryotic (viral and cellular) DNA polymerases previously proposed as Exo I segment 1, did not align with the Exo I segment of Escherichia coli DNA polymerase I (PolI)-like and protein-primed DNA polymerases; instead, a new conserved region of aa similarity was identified in T4 DNA polymerase and eukaryotic (viral and cellular) DNA polymerases as their corresponding Exo I segment. Therefore, according to our alignment, the recently reported T4 DNA polymerase site-directed mutants, D189A and E191A [Reha-Krantz et al., Proc. Natl. Acad. Sci. USA 88 (1991) 2417-2421], do not correspond to what we now consider the critical Exo I motif of PolI. As discussed in this communication, the functional importance of conserved segments Exo I, Exo II and Exo III is supported by site-directed mutagenesis in PolI, and in phi 29, T7 and delta(Sc) DNA polymerases. Furthermore, genetically selected T4 DNA polymerase mutator mutants form two main clusters, centered in the conserved segment Exo III and in the newly identified Exo I segment.     

Abolition of Intrinsically Bent DNA Structure Components in AT Clusters by Netropsin Interaction; Titration Viscometric Investigations

Abstract

It is argued that the enhancement of the apparent DNA contour length by the specifically binding non-intercalating drug netropsin (Nt) (Reinert et al., NAR 9,2335, 1981) at very low Nt/DNA-phosphate ratios essentially is the result of an abolition of periodically arranged intrinsic helix bends in A · T rich tracts of base pairs.

In the preceding paper the existence of pronounced DNA tertiary structure components has been postulated for (two species of) natural eukaryotic DNA. The resulting model suggests local apparent solenoid-related DNA tertiary structure components at high sodium ion concentration c_s, partly/totally molten out at 45/60 C. With decreasing c_s the tertiary structure components have been found to be gradually reduced, at least below c_s = 0.010 M, as titration viscometrically revealed by a gradual rise of the apparent DNA contour length (Reinert et al., JBSD 9, 537, 1991).

Hence, we performed titration viscometric analyses about Nt interaction with calf thymus DNA (ctDNA) at c_s = 0.075 M, 0.010 M and 0.004 M Na⁺. The concomitant DNA conformational changes are quantitatively described in terms of the relative changes of both DNA persistence length and hydrodynamically operative apparent DNA contour length for the three first resolved interaction modes below a Nt/DNA-P ratio of 0.03.

These experiments, together with previous respective analyses at c_s = 0.20 M Na⁺ and different temperatures (I.e.), suggest that those DNA sites binding Nt most strongly predominantly are responsible for the formation of solenoid-related DNA tertiary structure components. Most probably these are A tract-containing sequences. As the essential factor for their apparent elongation effect at low Na⁺ concentrations, a gradual alteration of the number of base pairs per helix turn seems to occur below c_s = 0.010 M Na⁺ and, concomitantly, a change in phasing between intrinsic helix bends and helix screw.     

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.     

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin

The interaction between the B-form specific ligands netropsin (Nt) and distamycin-3 (Dst-3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non-B DNA form, putatively a Z-like form. Three polymers with strict alternating purine-pyrimidine sequences and GC content from 100-0% have been tested: poly(dG-dC) . poly(dG-dC), poly(dA-dC) . poly(dG-dT) and poly(dA-dT) . poly(dA-dT). The titrations by Nt and Dst-3 were followed by circular dichroism. Although specific binding of Nt to the Z-form of poly(dG-dC) . poly(dG-dC) does not occur, Nt reverses this Z structure to the B-type conformation; Dst-3 is, however, totally inefficient. The presumed non-B or Z-like structure of poly(dA-dC) . poly(dG-dT) is reversed to the B-form upon interaction with Nt; Dst-3 also induces this reversal but at higher ligand ratios. The modified B-structure of poly(dA-dT) . poly(dA-dT) in low water activity is efficiently reversed to the B-form by interaction with both Nt and Dst-3.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.     

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5′GCTCTTC N1/N4 3′. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).     

An endonucleolytic activity of nuclease TT1 specific for superhelical DNA.

Highly purified nuclease TT1 from T. thermophilus HB8 acts on a linear single- and double-stranded DNA as an exonuclease and produces 5'-mononucleotides either from the 5'- or 3'-terminus. It was found that the enzyme also possesses an endonuclease activity specific for superhelical (form I) and single-stranded circular DNA. Form I of various kinds of DNA (phi X174, PM2, Co1E1 and RF 1010 etc.) is nicked to yield first relaxed circles (form II) and then nicked at the opposite site to yield unit length linear DNA (form III), which is subsequently hydrolyzed from the 5'- or 3'-terminus. A single cleavage of the form I of phi X174 DNA seemed to occur at a limited number of unique sites. Both endonuclease and the known exonuclease activities co-migrate on polyacrylmide gels, show the same pH and temperature optima, are stimulated by Mg2+ and are inactivated by EDTA similarly.     

In vivo restriction. Sequence and structure of endonuclease II-dependent cleavage sites in bacteriophage T4 DNA.

Endonuclease II of bacteriophage T4 is required for in vivo restriction of cytosine-containing DNA from its host, Escherichia coli, (as well as from phage mutants lacking cytosine modification), normally the first step in the reutilization of host DNA nucleotides for synthesis of phage DNA in infected cells. The phage cytosine-DNA is fragmented incompletely to yield genetically defined fragments. This restriction is different from that of type I, II, or III restriction enzymes. We have located seven major endonuclease II-dependent restriction sites in the T4 genome, of which three were analyzed in detail; in addition, abundant sites were cleaved in less than or equal to 5% of all molecules. Sites I, II, and III shared the sequence 5'-CCGNNTTGGC-3' and were cleaved in about 25% (I and III) and 65% (II) of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base. The less frequently cleaved sites I and III deviated from site II in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand. Thus, interaction with a particular helical structure as well as recognition of the bases in DNA appears important for efficient cleavage.     

Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.

The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene. Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities. Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants. Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II. A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding. However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity. This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

E. coli DNA polymerase. A study of the mechanism of primer binding using oligothymidylate analogs with ethylated internucleotide phosphate groups

The following individual diastereomers of oligothymidylate ethyl esters (the alkyl phosphodiester group is asymmetric with R or S configuration) have been prepared: d[(Tr)8Tp'(Et)T] (I), d[(Tp)8Tp'(Et)T] (II), d[(Tp)8Tp'(Et)TpT] (III), d[(Tp)8Tp' X (Et)TpT] (IV). A totally esterified analogue d[[(Tp(Et)7]T] (V) was obtained as a diastereomeric mixture. All oligothymidylate derivatives revealed substrate activity as primers of DNA polymerase with poly(dA) as a template. The values of the maximal reaction rates were equal to 14; 2,6; 68; 24 and 0,1% for oligothymidylates (I)-(V) with respect to Vm value (100%) for (Tp)9T. Km values of oligothymidylates (I)-(V), 2,7; 2,5; 0,51; 7,2 microM, were obtained in relation to Km for d[(Tp)9T] (0,4 microM). Diastereomers (I) and (II) were not destroyed by Klenow fragment of DNA polymerase I which has only 3'----5' exonuclease activity. However, these derivatives were hydrolyzed by complete DNA polymerase I due to its 5'----3' exonuclease activity, the reaction rate being 3-10 times lower than in case of d[(Tp)9T]. The data suggest an essential contribution to the primer binding from the positive enzyme group interaction with the 3'-end negatively charged phosphate group of oligonucleotide, together with the primer complementary interaction with the template. At least two phosphodiester groups of the oligonucleotide primer are essential for the reaction of polymerization following the correct binding.