Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.     

NaeI endonuclease binding to pBR322 DNA induces looping.

Previous work has demonstrated the existence of both resistant and cleavable NaeI sites. Cleavable sites introduced on exogenous DNA can act in trans to increase the catalysis of NaeI endonuclease cleavage at resistant sites without affecting the apparent binding affinity of the enzyme for the resistant site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. This activation suggests allosteric regulation of NaeI cleavage by distant cis- and trans-acting sites in DNAs containing both resistant and cleavable sites. Plasmid pBR322 contains four NaeI sites, at least one of which is resistant to cleavage. Electron microscopy is used here to demonstrate that NaeI endonuclease simultaneously binds to multiple recognition sites in pBR322 DNA to form loops with NaeI protein bound at the loop's base. The maximum number of loops formed with a common base suggests four binding sites per enzyme molecule. Looping was inhibited by addition of enzyme-saturating amounts of double-stranded oligonucleotide containing an NaeI site, whereas another double-strand oligonucleotide without the NaeI site had no effect. The number of loops seen was not above background when double-stranded M13 DNA, which contains only a single NaeI recognition site, was used as substrate.     

Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species.

Previous work has described the novel ability to modulate in vitro the activity of restriction endonuclease NaeI from Nocardia aerocoligenes by using cleavable DNA and spermidine [Conrad & Topal (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. In this paper we report the results of a study of 49 type II restriction enzymes from a variety of bacterial species. On the basis of the rates of cleavage observed, we found that in addition to expected cleavable sites a number of enzymes had slow and resistant cognate recognition sites. Resistant sites were identified for BspMI, NaeI, and NarI; slow sites were identified for HpaII, NaeI, and SacII. Cleavage of these sites was found to be significantly enhanced by the addition of cleavable DNA or spermidine. We demonstrate that for BspMI, as for NaeI, activator DNAs increased Vmax without altering Km, whereas for HpaII, NarI, and SacII activator DNAs decreased Km without changing Vmax. Comparison among the Kms for NaeI cleavage of several different substrates demonstrated that distant DNA sequences can affect DNA recognition by the activated enzyme. Our observations extend DNA activation of the Nocardia NaeI endonuclease to restriction endonucleases from Nocardia argentinensis (NarI), Bacillus species M (BspMI), Haemophilus parainfluenza (HpaII), and Streptomyces achromogenes (SacII). In addition, activation has now been found to affect slow as well as resistant recognition sites.     

DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites

Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis.     

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB–CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB–CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.     

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.     

Investigations into the sequence-selective binding of mithramycin and related ligands to DNA.

The preferred binding sites for mithramycin on four different DNA fragments have been investigated by DNAase I footprinting. Sites containing at least two contiguous GC base pairs are protected by the antibiotic, the preferred binding site consisting of the dinucleotide step GpG (or CpC). Related antibiotics chromomycin and olivomycin produce similar, but not identical footprinting patterns suggesting that they can recognize other sequences as well. All three antibiotics induce enhanced rates of enzyme cleavage at regions flanking some of their binding sites. These effects are generally observed in runs of A and T and are attributed to DNA structural variations induced in the vicinity of the ligand binding site. The reaction of dimethylsulphate with N7 of guanine was modified by the presence of mithramycin so that we cannot exclude the possibility that these antibiotics bind to DNA via the major groove.     

A consensus DNA-binding site for the androgen receptor.

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.     

Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.     

Enthalpy/entropy compensation: influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes

The effect of the context of the flanking sequence on ligand binding to DNA oligonucleotides that contain consensus binding sites was investigated for the binding of the intercalator 7-amino actinomycin D. Seven self-complementary DNA oligomers each containing a centrally located primary binding site, 5'-A-G-C-T-3', flanked on either side by the sequences (AT)(n) or (AA)(n) (with n = 2, 3, 4) and AA(AT)(2), were studied. For different flanking sequences, (AA)(n)-series or (AT)(n)-series, differential fluorescence enhancements of the ligand due to binding were observed. Thermodynamic studies indicated that the flanking sequences not only affected DNA stability and secondary structure but also modulated ligand binding to the primary binding site. The magnitude of the ligand binding affinity to the primary site was inversely related to the sequence dependent stability. The enthalpy of ligand binding was directly measured by isothermal titration calorimetry, and this made it possible to parse the binding free energy into its energetic and entropic terms. Our results reveal a pronounced enthalpy-entropy compensation for 7-amino actinomycin D binding to this family of oligonucleotides and suggest that the DNA sequences flanking the primary binding site can strongly influence ligand recognition of specific sites on target DNA molecules.     

The activity of the EcoRV restriction endonuclease is influenced by flanking DNA sequences both inside and outside the DNA-protein complex.

The EcoRV restriction endonuclease cleaves DNA not only at its recognition sequence but also at most other sequences that differ from the recognition site by one base pair. Compared to the reaction at the recognition site, the reactions at noncognate sites are slow but 1 out of the 12 noncognate sites on the plasmid pAT153 is cleaved more than 50 times faster than any other. The increase in the reaction rate at the preferred noncognate site, relative to other sites, was caused by the DNA sequences in the 4 base pairs from either side of the site. For enhanced activity by EcoRV, particular bases were needed immediately adjacent to the site, inside the DNA-protein complex. At these loci, the protein interacts with the phosphate groups in the DNA and the flanking sequence may control the activity of the enzyme by determining the conformation of the DNA, thus aligning the phosphate contacts. But the preferential cleavage also depended on sequences further away from the site, at loci outside the complex. At external positions, beyond the reach of the protein, the EcoRV enzyme required flanking sequences that give rise to flexibility in DNA conformation. These may facilitate the distortion of the DNA required for catalysis by EcoRV.