Purine Tracts Research Articles

New insights into calicheamicin-DNA interactions derived from a model nucleosome system

Using the Xenopus borealis 5S RNA gene, we have identified several new features of the interaction of calicheamicin (CAL), an enediyne antitumor agent, with nucleosomal and naked DNA targets. CAL-mediated DNA damage was generally reduced by incorporation of the DNA into a nucleosome. However, in one instance, the frequency of DNA damage was enhanced in the nucleosome compared to naked DNA. This increase in CAL damage may result from bending-induced changes in the target site, while the association of histone proteins with DNA in the nucleosome may generally reduce the affinity of CAL for its targets by imposing dynamic constraints on the DNA, by altering target structure, or by steric hindrance. One implication of these observations is that new structural features created by incorporation of DNA into chromatin may produce ‘hot spots’ for CAL-mediated DNA damage not apparent in naked DNA studies. In a second set of experiments, the orientation of CAL at damage sites in naked 5S rDNA was determined. The results suggest that minor groove width per se is not a major determinant of CAL target selection. Our studies support the generality of an oligopurine recognition element, with the additional requirement that the purine tract is interrupted at the 3′-end by a pyrimidine(s). To account for these observations, we propose a model in which CAL recognizes the unique structural and dynamic features associated with the 3′-end of an oligopurine tract. Finally, we conclude that the dyad axis of pseudosymmetry of the 5S rRNA gene nucleosome cannot be determined with any degree of certainty. This places significant limitations on the interpretation of results from the study of drug-DNA interactions with reconstituted nucleosomes.

Sequence Composition Effects on the Energetics of Triple Helix Formation by Oligonucleotides Containing a Designed Mimic of Protonated Cytosine

A nonnatural nucleoside, 1- (2-deoxy-β-ribofuranosyl)-3-methyl-5-amilHn-op-y razol0[4,3-d]pyrimidin-7-one (P), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine.purine.pyrimidine triple helix. Quantitative footprint titration experiments at neutral pH (22 °C, 100 mM NaCl, 10 mM bis-tris, 250 μM spermine) reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing P or 5-methylcytosine (^mC). Purine tracts of sequence composition 5'-d(AAAAAGAGAGAGAGA)-3' are bound by oligonucleotide 5'-d (TTTTT^mCT^mCT^mCT^mCT^mCT)-3' 4 orders of magnitude more strongly than by 5'-d (TTTTTPTPTPTPTPT)-3' (K_T ≈ 3 x l0^9 M^(-1) and KT = 1 x l0^5 M^(-1), respectively). Conversely, purine tracts of sequence composition 5'-d(AAAAGAAAAGGGGGGA)-3' are bound by oligonucleotide 5'-d(TTTT^mCTTTT)-3' 5 orders of magnitude less strongly than by 5'-d(TTTT^mCTTTTPPPPPPT)-3' (K_T < 5 x l0^4 M^(-1) and K_T ≈ 4 x l0^9 M^(-1), respectively). The complementary nature of P and ^mC expands the repertoire of G-rich sequences which may be targeted by triple helix formation.

6-Oxocytidine a novel protonated C-base analogue for stable triple helix formation.

2'-O-Methyl-3'-O-phosphoramidite building blocks of 6-oxocytidine 6 and its 5-methyl derivative 7, respectively, were synthesized and incorporated via phosphoramidite chemistry in 15 mer oligodeoxynucleotides [d(T72T7), S2; d(T73T7), S3] to obtain potential Py.Pu.Py triplex forming homopyrimidine strands. UV thermal denaturation studies and CD spectroscopy of 1:1 mixtures of these oligomers and a 21 mer target duplex [d(C3A7GA7C3)-d(G3T7CT7G3), D1] with a complementary purine tract showed a nearly pH-independent (6.0-8.0) triple helix formation with melting temperatures of 21-19 degrees C and 18.5-17.5 degrees C, respectively (buffer system: 50 mM sodium cacodylate, 100 mM NaCl, 20 mM MgCl2). In contrast, with the corresponding 15mer deoxy-C-containing oligonucleotide [d(T(7)1T7), S1] triplex formation was observed only below pH 6.6. Specificity for the recognition of Watson-Crick GC-base pairs was observed by pairing the modified C-bases of the 15mers with all other possible Watson-Crick-base compositions in the target duplex [d(C3A7XA7C3)-d(G3T7YT7G3), X = A,C,T; Y = T,G,A, D2-4]. Additionally, the Watson-Crick-pairing of the modified oligomers S2 and S3 was studied.

RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination.

Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination. We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions. In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments. We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels. These altered mobilities are resistant to RNase A but sensitive to RNase H. Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA. Importantly, complex formation only occurs upon transcription in the physiologic orientation. Reaban and Griffin [1990 Nature, 348, 342-344] found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment. Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand. These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.

Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA.

The genome of all retroviruses consists in two homologous RNA molecules associated near their 5' end in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for several functions of the retroviral cycle such as encapsidation, reverse transcription, and translation. In human immunodeficiency virus type 1 (HIV-1), a region downstream of the splice donor site was initially postulated to mediate dimerization. However, we recently showed that the dimerization initiation site is located upstream of the splice donor site and suggested that dimerization may initiate through a loop-loop interaction. Here, we show that single base mutations in the palindromic loop of the dimerization initiation site completely abolish dimerization, while introduction of compensatory mutations restores the process. Furthermore, two single nucleotide mutants that are unable to form homodimers efficiently codimerize, while the wild type RNA and the compensatory mutant, which both form homodimers, are unable to codimerize. These results unambiguously prove the interaction between the palindromic loops of each monomer. By contrast, none of the deletions that we introduced downstream of the splice donor site abolishes dimerization. However, deletions of two purine tracts located in the vicinity of the initiation codon of the gag gene significantly decrease the thermal stability of the HIV-1 RNA dimer.

Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides 1-707 of HIV-1 RNA. These nucleotides map exclusively in a restricted area upstream of the splice donor site and downstream of the primer binding site. They are centered around a palindromic sequence (GUGCAC279) located in a hairpin loop. Our results support a model in which dimer formation is initiated by the annealing of the palindromic sequences, possibly by a loop-loop interaction between the two monomers. Further experiments show that the deletion of the stem-loop or base substitutions in the loop abolish dimerization, despite the presence of the previously postulated dimer linkage structure. On the other hand, deletions of the purine tracts downstream of the splice donor site do not prevent dimerization. Therefore, we conclude that the palindromic region represents the dimerization initiation site of genomic RNA.

Specific recognition of CG base pairs by 2-deoxynebularine within the purine.purine.pyrimidine triple-helix motif.

The sequence-specific recognition of double-helical DNA by oligodeoxyribonucleotide-directed triple-helix formation is limited mostly to purine tracts. Within the geometric constraints of the phosphate-deoxyribose position of a purine-purine-pyrimidine triple-helical structure, model building studies suggested that the deoxyribonucleoside 2'-deoxynebularine (dN) might form one specific hydrogen bond with cytosine (C) or adenine (A) of Watson-Crick cytosine-guanine (CG) or adenine-thymine (AT) base pairs. 2-Deoxynebularine (dN) was incorporated by automated methods into purine-rich oligodeoxyribonucleotides. From affinity cleavage analysis, the stabilities of base triplets within a purine.purine.pyrimidine (Pu.Pu.Py) triple helix were found to decrease in the order N.CG approximately N.AT >> N.GC approximately N.TA (pH 7.4, 37 degrees C). Oligodeoxyribonucleotides containing two N residues were shown to bind specifically within plasmid DNA a single 15 base pair site of the human immunodeficiency virus genome containing two CG base pairs within a purine tract. This binding event occurs under physiologically relevant pH and temperature (pH 7.4, 37 degrees C) and demonstrates the utility of the new base. Quantitative affinity cleavage titration reveals that, in the particular sequence studied, an N.CG base triplet interaction results in a stabilization of the local triple-helical structure by 1 kcal.mol-1 (10 mM NaCl, 1 mM spermine tetrahydrochloride, 50 mM Tris-acetate, pH 7.4, 4 degrees C) compared to an A.CG base triplet mismatch.

Recognition of all four base pairs of double-helical DNA by triple-helix formation: design of nonnatural deoxyribonucleosides for pyrimidine.cntdot.purine base pair binding

The sequence-specific recognition of double-helical DNA by oligonucleotide-directed triple-helix formation is limited mostly to purine tracts. Design leads that could expand the recognition code to all four Watson-Crick base pairs would provide one step toward a general solution targeting single sites in megabase size DNA. The nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamidophenyl)imidazole (D3) was synthesized in four steps and incorporated by automated methods into pyrimidine oligodeoxyribonucleotides. Within a pyrimidine oligonucleotide, D3 binds pyrimidine.purine base pairs with higher affinity than it binds purine.pyrimidine base pairs. From affinity-cleaving analysis, the stabilities of base triplets decrease in the order D3.TA is similar to D3.CG > D3.AT > D3.GC. Such specificity allows binding by triple-helix formation at an 18 base pair site in SV40 DNA containing all four base pairs at physiologically relevant pH and temperature. The stabilities of these novel triplets may be an example of shape-selective recognition of CG and TA Watson-Crick base pairs in the major groove.

Static and statistical bending of DNA evaluated by Monte Carlo simulations.

To investigate the influence of thermal fluctuations on DNA curvature the Metropolis procedure at 300 K was applied to B-DNA decamers containing A5.T5 and A4.T4 blocks. Monte Carlo simulations have confirmed the DNA bending anisotropy: B-DNA bends most easily in a groove direction (roll). The A5.T5 block is more rigid than the other sequences; the pyrimidine-purine dimers are found to be the most flexible. For A5TCTCT, A5CTCTC, and A5GAGAG, the average bend angle per decamer is 20-25 degrees in a direction toward the minor groove in the center of the A5.T5 tract, which is consistent with both the "junction" and "wedge AA" models. However, in A5T5, A4T4CG, and T4A4GC, bending is directed into the grooves at the 5' and 3' ends of purine tracts. Thus, directionality of bending caused by An.Tn blocks strongly depends on their neighboring sequences. These calculations demonstrate that the sequence-dependent variation of the minor-groove width mimics the observed hydroxyl radical cleavage pattern. To estimate the effect of fluctuations on the overall shape of curved DNA fragments, longer pieces of DNA (up to 200 base pairs) were generated. For sequences with strong curvature (A5X5 and A4T4CG), the static model and Monte Carlo ensemble give similar results but, for moderately and slightly curved sequences (A5T5 or T4A4GC), the static model predicts a much smaller degree of bending than does the statistical representation. Considering fluctuations is important for quantitative interpretation of the gel electrophoresis measurements of DNA curvature, where both the static and statistical bends are operative.

Recognition of mixed-sequence duplex DNA by alternate-strand triple-helix formation

Oligodeoxyribonucleotide-directed triple-helix formation offers a chemical approach for the sequence-specific binding of double-helical DNA that is 10^6 times more specific than restriction enzymes. Because triple-helix formation by pyrimidine oligonucleotides is limited to purine tracts, it is desirable to find a general solution whereby oligonucleotides could be used to bind all four base pairs of intact duplex DNA (37 °C, pH 7.0). Approaches toward such a goal include the following: the search for other natural triplet specificities, such as G-TA triplets; the design of nonnatural bases for completion of the triplet code; the incorporation of abasic residues for nonreading of certain base pairs; and the design of oligonucleotides capable of binding alternate strands of duplex DNA by triple-helix formation. We report that a pyrimidine oligodeoxyribonucleotide-EDTA-Fe containing a 3'-3' phosphodiester and a 1,2-dideoxy-~ribose linker binds and cleaves a mixed-sequence double-helical DNA target site by alternate-strand triple-helix formation.

Triplex DNA in plasmids and chromosomes

Circular plasmids containing pyrimidine · purine tracts can form both inter- and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC) 45 · (GA) 45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4. plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G) n · (C) n and (TCC) n · (GGA) n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

Complete nucleotide sequence of the gene for human interleukin 1 alpha.

The chromosomal gene for human interleukin 1 alpha (IL-1 alpha) was isolated from a human genomic DNA library by using as a probe cloned human IL-1 alpha cDNA. Complete nucleotide sequence of about 12 kilobase pairs (kbp) long was determined and the structure and organization of this gene were elucidated. This gene contains seven exons and six introns. The first exon encodes the 5'-untranslated region. Most of the prepeptide portion of the precursor polypeptide is encoded by the next three exons, and the mature form of IL-1 alpha is encoded by the remaining three exons. The last exon also encodes the intronless 3'-untranslated region. The fourth intron as well as both 5'- and 3'-flanking regions contains the sequence of an Alu family member. In the middle part of the last intron, a 46-bp sequence with a unique structure is repeated five times in a head to tail manner. These repeats are flanked by the regions containing alternative purine and pyrimidine tracts. In the 5'-flanking region, immediately upstream of the putative TATA box, the 16-bp sequence highly homologous to the binding site of the adenovirus 2 major late promoter transcription factor is identified. The nucleotide sequence reported here showed only one nucleotide substitution in the 3'-untranslated exon in comparison with the nucleotide sequence of the cDNA from HL-60, a promyelocytic leukemia cell line, previously cloned and sequenced in our laboratories.

Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH.

Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes.

Specific binding site of E. coli initiation factor 3 (IF3) at a 3′-terminal region of MS2 RNA

INITIATION factor 3 (IF3) and ribosomal protein S1 have been shown to be essential in E. coli for the binding of the ribosome to natural mRNA. Both proteins bind to the 30S ribosomal subparticle at sites close to the 3′-terminus of 16S rRNA (refs 1, 2) and S1 protein interacts specifically with 16S rRNA at the pyrimidine-rich sequence ACCUCCU (ref. 3). The role of S1 protein may be to open up a doublestranded structure at this terminus3 thereby allowing base pairing to a purine tract in the mRNA near to the initiator triplet4,5 and it has been suggested that IF3 stabilises this annealing6. Such base pairing, according to current theory4–6, is the basis of the specific binding of the 30S subparticle to the initiation sites in mRNA. To elucidate the mechanism of this highly specific interaction, it is important to know whether the proteins involved in the binding contribute to the recognition of specific sites in the mRNA molecule. An approach to this problem is to look for sequence-specific interactions with mRNA. Both proteins can bind directly to mRNA (refs 7–10), but the specificity of binding has been investigated only in the case of S1 protein. No sequence specificity has been observed in the binding of this protein to R17 RNA (ref. 10). In Qβ RNA, however, a specific binding site has been identified near the 3′ end of the molecule10. We have investigated whether the binding of IF3 to MS2 RNA (which is very closely related to R17 RNA) is sequence specific and report here that IF3 binds specifically and reproducibly to MS2 RNA at a sequence in the untranslated region near to the 3′-terminus of the molecule.

The structure of the DNA of bacteriophage φX174: VI. Pyrimidine sequences in the complementary strand of the replicative form

Partially synthetic duplex circles of φX174 am3 DNA, specifically labeled in the pyrimidines of the complementary strand, were prepared in vitro using Escherichia coli DNA polymerase and a template of φX DNA. This material was degraded to pyrimidine tracts which were dephosphorylated and separated into fractions of uniform length by chromatography on DEAE-Sephadex. Chain lengths were assigned to the various isopliths by comparison with the profile obtained from labeled viral DNA tracts. By a combination of column Chromatographic and mapping techniques, a complete catalog of the pyrimidine tracts in the complementary strand of φX replicative form DNA (and thus, by complementarity, of the purine tracts of φX viral DNA) has been compiled.

Ribosome degradation and the degradation products in starved Escherichia coli: II. Changes in base sequence of ribosomal RNA during degradation induced by phosphate and magnesium starvation

Changes in the base sequence of ribosomal RNA isolated from ribosomes in phosphate- or Mg 2+-deficient Escherichia coli were investigated. The frequency of purine tracts differed to various extents from that of rRNA obtained from normal cells. The purine tract distribution was determined by ribonuclease digestion followed by separation of the products by DEAE-cellulose chromatography. This difference from normal rRNA sequence was greater in the phosphate-deficient ribosomes from the wild strain than it was in those of Mg 2+-deficient wild stain or the phosphate deficient ribonuclease I-less mutant strain, Q-13. The size of the RNA's in intact ribosomes in these deficient states was normal as judged by sedimentation analysis and the total base composition of rRNA was slightly different from that in normal cells. The implication of the results is discussed proposing two possible hypothesis.

Structure of the DNA of bacteriophage ΦX174: V. Purine sequences

Methods are described for the preparation of DNA, specifically labeled with carbon-14 in the purines, from bacteriophage ΦX174. This DNA, mixed with unlabeled calf thymus DNA as a carrier, has been degraded to purine tracts by hydrazinolysis and subsequent alkaline hydrolysis. The purine tracts have been separated according to magnitude by chromatography on a column of DEAE-Sephadex, at 55°C in the presence of 7 M -urea. The proportions of purines from calf thymus DNA, found in tracts of all magnitudes up to twelve, agree well with the previously measured proportions of pyrimidines in corresponding tracts, as would be expected from the complementary DNA structure. The complementarity restriction does not apply to ΦX DNA, and there appear to be purine tracts of greater length than any pyrimidine tract. A significant discrepancy has appeared between the numbers of purine and pyrimidine tracts, particularly between the numbers of solitary adenines and thymines. This may indicate an unsuspected flaw in these methods or an unusual feature in the structure of ΦX DNA.