Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

Nucleosomes will not form on double-stranded RNa or over poly(dA).poly(dT) tracts in recombinant DNA.

We have been unable to "force" double-stranded RNA to fold into nucleosome-like structures using several different histone-RNA "reconstitution" procedures. Even if the histones are first stabilized in octameric form by dimethylsuberimidate cross-linking they are still unable to form specific complexes with the RNA. Moreover double-stranded RNA is unable to induce histones to assemble into octamers although we confirm that the non-nucleic acid homopolymer, polyglutamic acid, has this ability. We have also determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule. Thus nucleosomes cannot fold DNA containing an 80 base pair poly(dA).poly(dT) segment but a 20 base pair segment can be accommodated in nucleosomes fairly well. Segments of intermediate length can be accommodated but are clearly selected against. Poly(dA).poly(dT) differs only slightly from natural DNA in helix structure. Therefore either this homopolymer resists folding, or nucleosomes are very exacting in the nucleic acid steroid parameters they will tolerate. Such constraints may be relevant to nucleosome positioning in chromatin.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Long poly(A) tracts in the human genome are associated with the Alu family of repeated elements

Long poly(dA).poly(dT) tracts (poly(A) tracts), regions of DNA containing at least 20 contiguous dA residues on one strand and dT residues on the complementary strand, are found in about 2 X 10(4) copies interspersed throughout the human genome. Using poly(dA).poly(dA) as a hybridization probe, we identified recombinant lambda phage that contained inserts of human DNA with poly(A) tracts. Three such tracts have been characterized by restriction mapping and sequence analysis. One major poly(A) tract is present within each insert and is composed of from 28 to 35 A residues. In each case, the poly(A) tract directly abuts the 3' end of the human Alu element, indicating that the major class of poly(A) tracts in the human genome is associated with this family of repeats. The poly(A) tracts are also adjacent to A-rich sequences and, in one case, to a polypurine tract, having the structure GA3-GA3-GA4-GA6-GA5-GA4. We suggest that repetitive cycles of unequal crossing over may give rise to both the long poly(A) and polypurine tracts observed in this study.     

Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.

It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA).poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA).poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA).poly(dT) fragment of 10-20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.     

Structure of poly(dA).poly(dT) from data of x-ray diffraction, energy calculations and nuclear magnetic resonance

The results of X-ray diffraction studies of poly(dA).poly(dT) have been compared with the results of energy optimization and with the NMR data in solution. Slight refinement of the X-ray and energetically optimal models leads to a very good quantitative agreement with the NMR data, that suggests similarity of the poly(dA).poly(dT) structure in a condensed state and in solution. One of the features distinguishing these models from the classic B form is a narrowed minor groove of the double helix. The anomalous properties of DNA with this sequence can be related specific organization of the water molecules near the polynucleotide.     

Acoustical investigation of poly(dA).poly(dT), poly[d(A-T)], poly(A).poly(U) and DNA hydration in dilute aqueous solutions.

Apparent molar adiabatic compressibilities and apparent molar volumes of poly[d(A-T)].poly[d(A-T)], poly(dA).poly(dT), DNA and poly(A).poly(U) in aqueous solutions were determined at 1 degree C. The change of concentration increment of the ultrasonic velocity upon replacing counter ion Cs+ by the Mg2+ ion was also determined for these polymers. The following conclusions have been made: (1) the hydration of the double helix of poly(dA).poly(dT) is remarkably larger than that of other polynucleotides; (2) the hydration of the AT pair in the B-form DNA is larger than that of the GC pair; (3) the substitution of Cs+ for Mg2+ ions as counter ions results in a decrease of hydration of the system polynucleotide plus Mg2+, and (4) the magnitude of this dehydration depends on the nucleotide sequence; the following rule is true: the lesser is a polynucleotide hydration, the larger dehydration upon changing Cs+ for Mg2+ ions in the ionic atmosphere of polynucleotide.     

Anomalous structure and properties of poly (dA).poly(dT). Computer simulation of the polynucleotide structure with the spine of hydration in the minor groove.

The results of the search for low-energy conformations of poly(dA).poly(dT) and of the poly(dA).poly(dT) "complex" with the spine of hydration similar to that found by Dickerson and co-workers (Kopka, M.L., Fratini, A.V., Drew, H.R. and Dickerson, R.E. (1983) J. Mol. Biol. 163, 129-146) in the minor groove of the CGCGAATTCGCG crystals are described. It is shown that the existence of such a spine in the minor groove of poly(dA).poly(dT) is energetically favourable. Moreover, the spine of hydration makes the polynucleotide conformation similar to the poly(dA).poly(dT) structure in fibers and to the conformation of the central part of CGCGAATTCGCG in crystals; it also acquires features characteristic of the structure of poly(dA).poly(dT) and DNA oligo(dA)-tracts in solution. It is shown that the existence of the TpA step in conformations characteristic of the poly(dA).poly(dT) complex with the spine of hydration is energetically unfavourable (in contrast to the ApT step) and therefore this step should result in destabilization of the spine of hydration in the DNA minor groove. Thus, it appears that the spine of hydration as described by Dickerson and co-workers is unlikely to exist in the poly d(A-T).poly d(A-T) structure. The data obtained permit us to interpret a large body of experimental facts concerning the unusual structure and properties of poly(dA).poly(dT) and oligo(dA)-tracts in DNA both in fibers and in solution. The results provide evidence of the existence of the minor groove spine of hydration both in fibers and in solution on A/T tracts of DNA which do not contain the TpA step. The spine plays an active role in the formation of the anomalous conformation of these tracts.     

Nucleosome reconstitution on plasmid-inserted poly(dA) . poly(dT).

Chromatin was reconstituted from core histones and recombinant plasmid DNAs carrying poly(dA) . poly(dT) inserts of various lengths. A 97-bp insert was found to occupy discrete and regularly-spaced positions on the edges of the nucleosome. This insert cannot, however, be entirely included due to a block in the center of the particle. In contrast, nucleosomes reconstitute on a shorter 20-bp insert. In this case, the insert shows a marked preference for the edges of the particle. Possible structural and physiological implications of these observations are discussed.     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.     

A unique deletion in pBR322 DNA caused by insertion of poly(dA) · poly(dT)

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA) · poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA) · poly(dT) fragment of 10–20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction

X-ray diffraction in fibres revealed that the calcium salt of poly(dA).poly(dT) is a 10-fold double helix with a pitch of 3.23 nm. The opposite sugar-phosphate chains in the refined model are characterized by a complete conformational equivalence and contain sugars in a conformation close to C2'-endo. As a result a new model of the sodium salt of poly(dA).poly(dT) has been constructed, which is different from the Heteronomous DNA proposed earlier (S. Arnott et al., Nucl. Acids Res. 11, 4141 (1983)). The new model of Na-poly(dA).poly(dT) has conformationally similar opposite chains; it is a structure of the B-type, rather like that of Ca-poly(dA).poly(dT).