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.     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.     

Mechanism for N-acetyl-2-aminofluorene-induced frameshift mutagenesis by Escherichia coli DNA polymerase I (Klenow fragment)

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.     

Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Analysis of the binding of p53 to DNAs containing mismatched and bulged bases

The tumor suppressor protein p53 modulates cellular response to DNA damage by a variety of mechanisms that may include direct recognition of some forms of primary DNA damage. Linear 49-base pair duplex DNAs were constructed containing all possible single-base mismatches as well as a 3-cytosine bulge. Filter binding and gel retardation assays revealed that the affinity of p53 for a number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding conditions. However, other mismatches including G/T, which is bound strongly by hMSH2-hMSH6, were poorly recognized by p53. The general order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all the other mismatches.     

Strong structural effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

The NarI restriction enzyme recognition site, G1G2CG3CC, has been identified as a hotspot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF) on the basis of a forward mutation assay in plasmid pBR322 in the bacterium Escherichia coli. AAF binds primarily to the C-8 position of guanine residues, and the three guanines of the NarI site are similarly reactive. Despite this similar chemical reactivity, only binding of AAF to the G3 residue causes the -2 frameshift mutations. To study the mechanisms underlying the specificity of the mutagenic processing further, we monitored the structural changes induced by a single AAF adduct within the NarI site by means of CD spectroscopy and thermal denaturation. The NarI sequence was studied as part of the 12-mer ACCGGCGCCACA. The purification and characterization of the three isomers having a single AAF adduct covalently bound to one of the three guanines of this 12 mer are described. The analysis of the melting profiles of the duplexes formed when these three isomers are annealed with the oligonucleotide of complementary sequence shows the same destabilizing effect of the AAF adduct on the three DNA helices. It is also shown, from the CD spectra, that modification of guanine G1 or G2 by AAF does not induce major changes in the helical structure of DNA. On the other hand, modification of guanine G3 induces a change in the CD signal that suggests the formation of a local left handed structure within the 12-mer duplex. These results show the polymorphic nature of the DNA structure in the vicinity of an AAF adduct.     

Restriction enzyme mapping of carcinogen binding regions on pBR322.

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 phiX174 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.     

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.     

Restriction endonucleases HindII and TaqI cleave DNA with mismatched nucleotides within their recognition sequences.

Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.     

Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA.

An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 microM actinomycin. A chi(2) statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5'-(T/A)GC(A/T)-3', giving a p<0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258nM (average 98nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5'-TGCTGCT-3'), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiological conditions.     

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.     

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.     

The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

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.     

Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA

Many proteins involved in DNA repair systems interact with DNA that has structure altered from the typical B-form helix. Using magnetic beads to immobilize DNAs containing various types of structures, we evaluated the in vitro binding activities of two well-characterized DNA repair proteins, Escherichia coli MutS and human p53. E. coli MutS bound to double-stranded DNAs, with higher affinity for a G/T mismatch compared to a G/A mismatch and highest affinity for larger non-B-DNA structures. E. coli MutS bound best to DNA between pH 6 and 9. Experiments discriminated between modes of p53–DNA binding, and increasing ionic strength reduced p53 binding to nonspecific double-stranded DNA, but had minor effects on binding to consensus response sequences or single-stranded DNA. Compared to nonspecific DNA sequences, p53 bound with a higher affinity to mismatches and base insertions, while binding to various hairpin structures was similar to that observed to its consensus DNA sequence. For hairpins containing CTG repeats, the extent of p53 binding was proportional to the size of the repeat. In summary, using the flexibility of the magnetic bead separation assay we demonstrate that pH and ionic strength influence the binding of two DNA repair proteins to a variety of DNA structures.     

NMR evidence of the stabilisation by the carcinogen N-2-acetylaminofluorene of a frameshift mutagenesis intermediate.

Two heteroduplexes d(C1A2C3T4C5G6C7A8C9A10C11)-d (G12T13G14T15G16G17A18G19T20G21) containing a bulged guanine either unmodified or modified with the carcinogen N-2-acetylaminofluorene (AAF) have been studied by nuclear magnetic resonance (NMR) as models of slipped mutagenic intermediates (SMI). Conformational equilibria are observed in both the unmodified and the AAF-modified heteroduplexes. The major conformation of the unmodified duplex is one where the extra guanine is stacked in the helix and the major conformation of the AAF-modified heteroduplex is one where the AAF is external to the helix. Unusual sugar proton chemical shifts of C5- and G6-AAF indicate that the AAF ring is pointing out in the 5' direction. A strong increase in the modified heteroduplex melting temperature (+15 degrees C) is observed. Moreover, in contrast to the unmodified heteroduplex, which shows extensive melting in the vicinity of the bulged guanine, the base pairs around the bulge in the AAF-modified heteroduplex remain paired at temperatures up to 30 degrees C. This exceptional stability of the site around the bulged modified guanine is suggested to be responsible for the high rate of -1 mutation induced by AAF at repetitive sequences.     

Single-strand DNA binding of actinomycin D with a chromophore 2-amino to 2-hydroxyl substitution

A modified actinomycin D was prepared with a hydroxyl group that replaced the amino group at the chromophore 2-position, a substitution known to strongly reduce affinity for double-stranded DNA. Interactions of the modified drug on single-stranded DNAs of the defined sequence were investigated. Competition assays showed that 2-hydroxyactinomycin D has low affinity for two oligonucleotides that have high affinities (K(a) = 5-10 x 10(6) M(-1) oligomer) for 7-aminoactinomycin D and actinomycin D. Primer extension inhibition assays performed on several single-stranded DNA templates totaling around 1000 nt in length detected a single high affinity site for 2-hydroxyactinomycin D, while many high affinity binding sites of unmodified actinomycin D were found on the same templates. The sequence selectivity of 2-hydroxyactinomycin D binding is unusually high and approximates the selectivity of restriction endonucleases. Binding appears to require a complex structure, including residues well removed from the polymerase pause site.     

Stoichiometry and affinity for thymine DNA glycosylase binding to specific and nonspecific DNA

Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.     

Distamycin inhibition of topoisomerase I-DNA interaction: a mechanistic analysis.

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA).(dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was unessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable band. Alternative modes of inhibition are discussed.     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.