Restriction Enzyme Mapping of Carcinogen Binding Regions on pBR322

Abstract

Previous equilibrium binding experiments (S.A. Winkle and T.R. Krugh, Nucleic Acids Res. 9, 3175–3186 (1981)) suggested that the carcinogen N-hydroxy-N-acetyl-2-aminofluorene might exhibit preferential binding to a small number of sites on phiXl 74 DNA To examine whether the covalently binding analogue N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) also possesses high affinity sites, the plasmid pBR322 was reacted with 3H labeled acetoxyAAF to give one to sixteen adducts per DNA molecule. Thus only higher affinity sites would be affected. The DNA was subsequently cleaved with either Alu I, Hae III, Hha I, Hinf I or Hpa II restriction endonuclease and the restriction fragments isolated by gel electrophoresis. Examination of the distribution of 3H acetoxyAAF among the fragments was not random but, rather, with each enzyme, the acetoxyAAF was found predominantly in a few fragments. The locations of the bands containing the acetoxyAAF for each enzyme overlap - suggesting that there are regions on pBR 322 which contain high affinity sites for acetoxyAAF binding.     

Using lambda exonuclease inhibition assays to map carcinogen binding sites.

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.     

Locating binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene using restriction enzyme inhibition assays.

Restriction enzyme inhibition studies have been employed to map the locations of high affinity binding sites of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) on pBR322, phiX174 and SV40 DNAs. Bound carcinogen levels were kept low (less than 20 bound AAF moieties per DNA molecule) in order to observe only the binding to the high affinity sites. Inhibition of certain restriction enzymes was observed in a limited number of locations on these DNAs. Inhibition increased as bound AAF increased and the particular restriction enzymes inhibited varied with location. On all three DNAs, activities of these enzymes was not affected in other locations. Comparison of the sequences at the sites of inhibition on the three DNAs indicates that all sites have common sequence elements: the presence of either the sequence T(C/G)TT(G/C) or the sequence T(G/C)CTT(G/C).     

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.     

EcoRII can be activated to cleave refractory DNA recognition sites.

EcoRII restriction sites [5'-CC(A/T)GG] in phage T3 and T7 DNA are refractory to cleavage by EcoRII, but become sensitive to cleavage in the presence of DNAs which contain an abundance of EcoRII sensitive sites (e.g. pBR322 or lambda DNA). Studies using fragments of pBR322 containing different numbers of EcoRII sites show that the susceptibility to EcoRII cleavage is proportional to the number of sites in the individual fragment. We postulate that EcoRII is the prototype of restriction endonucleases which require at least 2 simultaneously bound substrate sites for their activation. EcoRII sites are refractory when they occur at relatively low frequency in the DNA. The restriction enzyme can be activated by DNA with a higher frequency of sites.     

Bsu2413I and Bfi2411I,two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification – Thermophilic endonucleases from two Bacillus species

Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified. The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I. The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp. The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases.     

Equilibrium binding of four anthracyclines to nucleic acids: thermodynamic properties and sequence selectivity.

The thermodynamic parameters of the interaction of the two anthracyclines 13-dihydrodaunomycin and marcellomycin with calf thymus DNA were examined by equilibrium binding studies. Enthalpy and entropy changes of the binding of both drugs show salt dependence profiles that cannot be rationalized by the polyelectrolyte theory. This feature is common to other anthracycline compounds. The nucleotide sequence binding preferences of daunomycin, adriamycin, 13-dihydrodaunomycin and marcellomycin have been studied by monitoring the degree of protection from cleavage by restriction endonucleases of linearized pBR322. Differential protection of pBR322 DNA against the cleavage of Bgl I and Ava II suggests that these drugs recognize changes in the sequences near the enzyme recognition site. Alterations of the electrophoretic restriction pattern of pBR322 in the presence of anthracyclines are dependent on time and on concentration. These results are discussed in relation to the existence of nucleotide sequences with different affinity for these drugs.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

Electron microscopic mapping of Thermus thermophilus RNA polymerase binding sites on plasmid pBR322

The binding of RNA polymerase from the extreme thermophile T. thermophilus HB8 to plasmid pBR322 was measured by electron microscopy. DNA-protein complexes were prepared at 35 and 60 degrees C. At both temperatures the enzyme binds strongly to sites which coincide with promoters P1, P2, P3 and P4 present in pBR322. At 60 degrees C, an additional binding site appears, which is located between P3 and P4. There is a high degree of correlation between RNA polymerase binding sites and the location of A-T rich regions on pBR322 DNA.     

pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long.

I have derived a complete restriction map of pBR322 from the total nucleotide sequence of the plasmid. Most of the restriction sites also have been demonstrated empirically. The exact sizes of all restriction fragments and the relative positions of the cuts are presented. These fragments can serve as accurate DNA size markers from small pieces up to the 4362 base pair length of pBR322. Inserts cloned in this vector may be characterized easily using this data.     

Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA.Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA).poly(dT) regions. The pentapeptide binds 6-7-base-pair sites with a preference for poly(dA).poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A + T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A + T rich binding site.     

SwaI, a unique restriction endonuclease from Staphylococcus warneri, which recognizes 5'-ATTTAAAT-3'.

A novel class-II restriction endonuclease designated SwaI was purified from Staphylococcus warneri. This enzyme cleaves adenovirus 2 DNA, SV40 DNA and M13mp7 at one site each, but does not cleave lambda, PhiX174, pBR322 or pBR328 DNA. SwaI recognizes the octanucleotide sequence 5'-ATTTAAAT-3', cleaving in the center of the recognition sequence creating blunt ended DNA fragments. SwaI was used to digest chromosomal DNA from various microorganisms and human cells.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

A sequence-specific endonuclease (Xmn I) from Xanthomonas manihotis.

A type II restriction endonuclease Xmn I with a novel site specificity has been isolated from Xanthomonas manihotis. Xmn I does not cleave SV40 DNA, but cleaves phi X174 DNA into three fragments, which constitute 76.61%, 18.08% and 5.31% of the total length of 5386 base pairs, and cleaves pBR322 DNA into two fragments of 55.71% and 44.29% of the entire 4362 base pairs. The nucleotide sequences around the cleavage sites made by Xmn I are not exactly homologous, but they have a common sequence of 5' GAANNNNTTC 3' according to a simple computer program analysis on nucleotide sequences of phi X174 DNA, pBR322 DNA and SV40 DNA. The results suggest that the cleavage site of Xmn I is located within its recognition sequence of 5' GAANNNNTTC 3'.     

Physical mapping of a 330 X 10(3)-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination.

Myxococcus xanthus was pulse-labeled with [3H]thymidine immediately after germination of dimethyl sulfoxide-induced spores. The restriction enzyme digests of the total chromosomal DNA from the pulse-labeled cells were analyzed by one-dimensional as well as two-dimensional agarose gel electrophoresis. Four PstI fragments preferentially labeled at a very early stage of germination were cloned into the unique PstI site of pBR322. By using these clones as probes, a restriction enzyme map was established covering approximately 6% of the total M. xanthus genome (330 X 10(3) base pairs). The distribution of the specific activities of the restriction fragments pulse-labeled after germination suggests a bidirectional mode of DNA replication from a fixed origin.     

pBR322DNA与8-甲氧补骨脂素光化学反应部位的碱基顺序特异性

用限制性核酸内切酶酶切试验研究了质粒pBR322 DNA经8-MOP及近紫外线作用后损伤部位的碱基顺序特异性。实验研究发现PUVA损伤的DNA在HindⅢ及RsaⅠ识别位置上酶切反应受到严重抑制,而在SphⅠ,EcoRⅠ,PvuⅡ,BamHI,PstⅠ识到位置上抑制轻微。通过对不同识别位置上碱基顺序及其光化学反应敏感性的分析,推断出DNA的TpA顺序可能是最易接受8-MOP光化学反应的部位。     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.