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The T antigen specified by SV40 virus is the only viral-encoded protein required for replication of SV40 DNA. T antigen has two activities that appear to be essential for viral DNA replication: specific binding to duplex DNA at the origin of replication and helicase activity that unwinds the two DNA strands. As judged by electron microscopy, DNA unwinding is initiated at the origin of replication and proceeds bidirectionally. Either linear or circular DNA molecules containing the origin of replication are effective substrates; with closed circular DNA, a topoisomerase capable of removing positive superhelical turns is required for an efficient reaction. Presence of an origin sequence on duplex DNA and a single-strand DNA-binding protein appear to be the only requirements for T antigen to catalyze unwinding. This reaction mediated by T antigen defines a likely pathway to precise initiation of DNA replication: (i) the sequence-specific binding activity locates the origin sequence, (ii) the duplex DNA is unwound at this site, and (iii) the DNA polymerase and primase begin DNA replication. A similar pathway has been inferred for the localized initiation of DNA replication by bacteriophage lambda and by Escherichia coli in which a sequence-specific binding protein locates the origin and directs the DnaB helicase to this site. Observations with the SV40 system indicate that localized initiation of duplex DNA replication may be similar for prokaryotes and eukaryotes.
In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.
We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.
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We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus. Within this region, MNase preferentially cleaved 140 sites. Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals. These clusters of preferential cleavage sites rarely occur within gene coding regions. The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG. Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand. An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence. The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern. Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content. Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability.
RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).
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Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.
A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.
Minimized potential energy calculations have been employed to locate and evaluate energetically a number of different models for DNA modified at carbon-8 of guanine by acetylaminofluorene (AAF). Three different duplex nonamer sequences were investigated. In addition to syn guanine models which have some denaturation and a Z-DNA model, we have found two new types of structures in which guanine remains syn and the AAF is placed in the minor groove of a B-DNA helix. One type features Hoogsteen base pairing between the modified guanine and protonated cytosine, with a sharply bent helix. The other (here termed the "wedge" model because the aromatic amine is wedged into the minor groove) maintains a single hydrogen bond between O6 of the modified guanine and N3 of protonated cytosine, with much less deformation of the helix, and close Van der Waals contacts between the AAF and the walls of the minor groove. Both types of structures (as well as the related forms produced by deprotonation of cytosine) are energetically important in all three sequences examined. The wedge-type model, which is most favored except in alternating G-C sequences, has been previously observed in a combined NMR and computational characterization of an aminofluorene (AF) modified guanine opposite adenine in a DNA duplex undecamer (D. Norman, P. Abuaf, B.E. Hingerty, D. Live, D. Grunberger, S. Broyde and D.J. Patel, Biochemistry 28, 7462 (1989)).
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Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.
We have synthesized and examined a number of fully and partly self-complementary palindromic oligodeoxynucleotides for their ability to assume in solution a unimolecular hairpin structure. The main results obtained by a combined optical and electrophoresis investigation show that: (i) DNA folding needs not be driven by mismatched base pairings over the dyad; fully self-complementary palindromic duplexes, comprising regular (CG)n DNA fragments, possess a considerable intrinsic propensity to make intramolecular base pairings; (ii) The duplex-hairpin interconversion is, in general, a slow process independent of the length and base composition of the palindrome; (iii) The palindromic sequences energetically least favored to form hairpin structures consist of C:G base pairs around the dyad axis and of T:A blocks in the arms of the inverted repeat; (IV) The base composition of the stem strongly influences the hairpin thermal stability. For instance, the substitution of one C:G with one A:T base pair in the stem helix of d(CG)7 diminishes the stability of the hairpin by 9 degrees C. It is found that the stability of the stem helix, in hairpins of defined sequence and with the same loop length, decreases in the order alternating-CG greater than homo-CG greater than AC(GT) greater than alternating-AT, i.e. as in polynucleotides. The thermodynamic parameters for the hairpin-coil transition are reported.
The enhancer-binding factor of human immunodeficiency virus (HIV)-1 was purified from human B cells by sequence-specific duplex oligonucleotide affinity chromatography. Gel retardation assay and footprint analysis showed that the purified factor bound specifically to the HIV-1 enhancer sequence, and protected both direct repeats in the HIV-1 enhancer. The purified factor consisted of four main polypeptides of the molecular weight of 36,000-42,000. At least three of them had enhancer-binding activity after elution from sodium dodecyl sulfate-polyacrylamide gel and renaturation. UV cross-linking analysis also showed that at least two of the polypeptides in purified fraction had a binding activity specific for the HIV-1 enhancer. The purified factor activated transcription from the HIV-1 promoter in vitro, confirming that it was indeed a transcription factor for HIV-1. The purified factor also recognized sequences in the immunoglobulin kappa gene enhancer and the major histocompatibility complex class I gene enhancer with almost the same affinity as the HIV-1 enhancer. These results suggested the existence of multiple proteins which recognize the kappa B-related sequences. This regulatory factor should help in the study of the biochemical pathway underlying HIV production from latently infected cells.
A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.