Target Base Pair Research Articles

Triplex formation involving 2',4'-BNA with 2-pyridone base analogue: Efficient and selective recognition of C:G interruption

We examined the thermodynamic properties of 2',4'-bridged nucleic acid containing 2-pyridone as a nucleobase (PB) to recognize a C interruption in the homopurine strand of the target duplex for pyrimidine motif triplex formation at neutral pH. The triplex formation involving triplex-forming oligonucleotide with PB is highly sequence-selective to specifically recognize C:G target base pair rather than the other G:C, T:A, or A:T base pairs. PB.C:G triad gives significantly larger binding constant than T.C:G triad, which has been known to be the most stable combination in natural base.C:G triad. Our results certainly support the idea that PB could be a key nucleoside to recognize a C interruption in the homopurine strand of the target duplex with high binding affinity and selectivity, and reduce the restriction of target sequences for triplex formation.

Bisulfite-induced cytosine deamination rates in E. coli SSB:DNA complexes

E. coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA). We used a lacZ α-complementation reversion assay to detect C→U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex. When incubated at 37°C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon. Across a 250 base pair target and over 23 scorable C→U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone. After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C→U events. The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations. The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.

Modeling multi-component protein-DNA complexes: the role of bending and dimerization in the complex of p53 dimers with DNA.

We used molecular modeling to study the optimal conformation of the complex between two p53 DNA-binding domain monomers and a 12 base-pair target DNA sequence. The complex was constructed using experimental data on the monomer binding conformation and a new approach to deform the target DNA sequence. Combined with an internal/helicoidal coordinate model of DNA, this approach enables us to bend the target sequence in a controlled way while respecting the contacts formed with each p53 monomer. The results show that the dimeric complex favors DNA bending towards the major groove at the dimer junction by a value close to experimental findings. In contrast to inferences from earlier models, the calculation of key contributions to the free energy of the complexes indicates a determinant role for DNA in the formation of the complex with the dimer of the p53 DNA-binding domains.

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog.

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

In vivo analysis of human T-cell leukemia virus type 1 reverse transcription accuracy.

Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZalpha peptide gene region (the mutational target). The vector was replicated by trans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 x 10(-6) mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

Structural effects of DNA sequence on T·A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T·A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)2 and the pyrrole counterpart (ImPyPyPy)2 bound to the six base-pair target site 5′-AGATCT-3′ in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 Å, respectively. The structures demonstrate that the principles of Hp/Py recognition of T·A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.

A search for base analogs to enhance third-strand binding to 'inverted' target base pairs of triplexes in the pyrimidine/parallel motif.

Eight base analogs were tested as third strand residues in otherwise homopyrimidine strands opposite each of the 'direct' (A.T and G.C) and 'inverted' (T.A and C.G) Watson-Crick base pairs, using UV melting profiles to assess triplex stability. The target duplexes contained 20 A.T base pairs and a central test base pair X.Y, while the third strand contained 20 T residues and a central Z test base. Z included 5-bromo-uracil, 5-propynyluracil, 5-propynylcytosine, 5-methyl-cytosine, 5-bromocytosine, hypoxanthine, 2-amino-purine and 2,6-diaminopurine. Some of the base analogs enhanced third strand binding to the target duplex with one or other 'inverted' central base pair relative to the binding afforded by any of the canonical bases. Other analogs did the same for the duplexes with the 'direct' target pairs. The increasing order of triplex stabilization by these base analogs is: opposite the 'inverted' base pairs, for T.A, A < C < 5-pC < 5-pU < T < 5-BrC < 5-meC < 5-BrU < 2-AP < 2,6-DAP < Hy < G, for C.G, 2-AP < A < Hy < G < 5-pC < 5-BrC < 5-meC < C < 2,6-DAP < T < 5-BrU < 5-pU; opposite the 'direct' base pairs, for A.T, 2-AP < A < 5-meC < C < G < Hy < 2,6-DAP < 5-pU < T = 5-BrU < 5-BrC < 5-pC, for G.C, G < 2,6-DAP < 2-AP < A < Hy < T < 5-BrU < 5-pU < 5-pC < 5-BrC < C < 5-meC.

Refined High-Field NMR Solution Structure of a Binary-Addressed Pyrene/Perfluoro-Azide Complementary DNA Oligonucleotide System Shows Extensive Distortion in the Central Nick Region

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and PyrpdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafluo'robenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-l-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 protonproton distance-range constraints, evaluated from the 1H-NOESY spectrum (Tmix=200rns, 600MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5°) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 20T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11Å to 24Å for the six final structures (17Å, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a ID variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.

A Comparison of H-Pin and Hairpin Polyamide Motifs for the Recognition of the Minor Groove of DNA

Solid-phase polyamide synthesis is extended to the H-pin motif using a Boc-protected bispyrrole monomer and bidirectional synthesis. Sequence-specific recognition of a designated seven base pair target site is achieved by the formation of a 10-ring H-pin polyamide⋅DNA complex in the minor groove. DNA binding properties are compared with the corresponding hairpin polyamides (see below).

Structures of HhaI methyltransferase complexed with substrates containing mismatches at the target base.

Three structures have been determined for complexes between HhaI methyltransferase (M.HhaI) and oligonucleotides containing a G:A, G:U or G:AP (AP = abasic or apurinic/apyrimidinic) mismatch at the target base pair. The mismatched adenine, uracil and abasic site are all flipped out of the DNA helix and located in the enzyme's active-site pocket, adopting the same conformation as in the flipped-out normal substrate. These results, particularly the flipped-out abasic deoxyribose sugar, provide insight into the mechanism of base flipping. If the process involves the protein pushing the base out of the helix, then the push must take place not on the base, but rather on the sugar-phosphate backbone. Thus rotation of the DNA backbone is probably the key to base flipping.

A Comparison of H-Pin and Hairpin Polyamide Motifs for the Recognition of the Minor Groove of DNA

Solid-phase polyamide synthesis is extended to the H-pin motif using a Boc-protected bispyrrole monomer and bidirectional synthesis. Sequence-specific recognition of a designated seven base pair target site is achieved by the formation of a 10-ring H-pin polyamide⋅DNA complex in the minor groove. DNA binding properties are compared with the corresponding hairpin polyamides (see below).

A novel DNA recognition mode by the NF-kappa B p65 homodimer.

The crystal structure of the NF-kappa B p65 (RelA) homodimer in complex with a DNA target has been determined to 2.4 A resolution. The two p65 subunits are not symmetrically disposed on the DNA target. The homodimer should optimally bind to a pseudo-palindromic nine base pair target with each subunit recognizing a 5'GGAA-3' half site separated by a central A-T base pair. However, one of the subunits (subunit B) encounters a half site of 5'-GAAA-3'. The single base-pair change from G-C to A-T results in highly unfavorable interactions between this half site and the base contacting protein residues in subunit B, which leads to an 18 degrees rotation of the N-terminal terminal domain from its normal conformation. Remarkably, subunit B retains all the interactions with the sugar phosphate backbone of the DNA target. This mode of interaction allows the NF-kappa B p65 homodimer to recognize DNA targets containing only one cognate half site. Differences in the sequence of the other half site provide variations in conformation and affinity of the complex.

Direct Real Time Observation of Base Flipping by theEcoRI DNA Methyltransferase

DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (KDDNA) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteady-state real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to k(methylation) determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.

Kinetic analysis of sequence-specific alkylation of DNA by pyrimidine oligodeoxyribonucleotide-directed triple-helix formation.

Attachment of a nondiffusible bromoacetyl electrophile to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence-specific alkylation of a guanine base in duplex DNA two base pairs to the 5'-side of a local triple-helical complex. Products resulting from reaction of 5'-ETTTTMeCTTTTMeCMeCTTTMeCTTTT-3' at 37 degrees C with a 29 base pair target duplex are determined by a gel mobility analysis to be oligonucleotides terminating in 5'- and 3' -phosphate functional groups, consistent with a mechanism involving alkylation, glycosidic bond cleavage, and base-promoted strand cleavage. The guanine-(linker)-oligonucleotide conjugate formed upon triple-helix-mediated alkylation at the N7 position of a guanine base in a 60 base pair duplex was identified by enzymatic phosphodiester hydrolysis of the alkylation products followed by reversed phase HPLC analysis. To determine the rate enhancement achieved by oligonucleotide-directed alkylation of duplex DNA, a comparison of rates of alkylation at N7 of guanine in double-stranded DNA by the N-bromoacetyloligonucleotide and 2-bromoacetamide was performed by a polyacrylamide gel assay. The reaction within the triple-helical complex on a restriction fragment was determined at 200 nM N-bromoacetyloligonucleotide to have a first-order rate constant k1 of (2.7 +/- 0.5) x 10(-5) S(-1) (t1/2 = 7.2 h). The reaction of 2-bromoacetamide with a 39 base pair duplex of sequence corresponding to the restriction fragment targeted by triple-helix formation was determined to have a second-order rate constant k2 of (3.6 +/- 0.3) x 10(-5) M(-1) S(-1). A comparison of the first-order and second-order rate constants for the unimolecular and bimolecular alkylation reactions provides an effective molarity of 0.8 M for bromoacetyl within the triple-helical complex.

The Mutation Rate of Human Immunodeficiency Virus Type 1 Is Influenced by thevprGene

A system has been designed to study thein vivoforward rate of mutation of human immunodeficiency virus type 1 (HIV-1) during one round of replication. A HIV-1 shuttle vector was used that contained thelacZα peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 × 10−5mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 reverse transcriptase with sense-strand RNA and DNA templates of thelacZα peptide gene in a cell-free system. To test the hypothesis that thevprgene product might, at least in part, account for the lower mutation rate observedin vivo,a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-typevprgene product. Avpr−shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of thevpr−vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence ofvpron the mutation rate. Replication of avpr−shuttle vector in the presence of a wild-typevprexpression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that thevprmutation could be complementedin trans.Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence ofvpron the mutation rate.

Solution Structure of a Pyrimidine·Purine·Pyrimidine Triplex Containing the Sequence-specific Intercalating Non-natural Base D 3

We have used NMR spectroscopy to study a pyrimidine·purine·pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-β-D-ribofuranosyl)- 4-(3-benzamido)phenylimidazole (D 3), in the third strand. The D 3base has been previously shown to specifically recognize T·A and C·G base-pairs viaintercalation on the 3′ side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D 3base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D 3base stack on the bases of all three strands and mimic a triplet. The conformation of the D 3base and its sequence specificity are discussed.

Forward mutation rate of human immunodeficiency virus type 1 in a T lymphoid cell line.

An in vivo assay was previously developed for detecting forward mutations in human immunodeficiency virus type 1 (HIV-1) in a single cycle of replication. This system uses the lacZalpha peptide gene as a reporter for mutations, and allows for the rates and types of mutations that occur to be determined. The forward mutation rate for HIV-1 in HeLa cells was found to be 3 x 10(-5) mutations per target base pair per cycle. To test whether the mutation rate was influenced by cell type, the mutation rate of HIV-1 in CEM-A cells, a T lymphoid cell line, was determined. The mutation rate of HIV-1 reverse transcription in CEM-A cells was found to be 4 x 10(-5) mutations per target base pair per cycle. The number and types of mutations observed were similar to that in HeLa cells. Specifically, base substitution mutations predominated, and G-to-A transition mutations were the most common base substitution. G-to-A hypermutants were also characterized. The difference in HIV-1 mutation rate between HeLa and CEM-A cells was not significant, indicating that the accuracy of HIV-1 reverse transcription is comparable in both the HeLa and CEM-A cell lines.

UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(C(+)-T)6:[d(A-G)6.d(C-T)6]; evidence for a proton switch.

Near-UV difference spectral analysis of the triplex formed from d(C-T)6 and d(A-G)6.d(C-T)6 in neutral and acidic solution shows that the third strand dC residues are protonated at pH 7.0, far above their intrinsic pKa. Additional support for ion-dipole interactions between the third strand dC residues and the G.C target base pairs comes from reduced positive dependence of triplet stability on ionic strength below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong positive dependence on hydrogen ion concentration. Molecular modeling (AMBER) of C:G.C and C+:G.C base triplets with the third strand base bound in the Hoogsteen geometry shows that only the C+:G.C triplet is energetically feasible. van't Hoff analysis of the melting of the triplex and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCl/0.005 M MgCl2 the enthalpy of melting (delta H degree obs) varies from 5.7 to 6.6 kcal.mol-1 for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.mol-1 for third strand dissociation in the triplex mixture. We have extended the condensation-screening theory of Manning to pH-dependent third strand binding. In this development we explicitly include the H+ contribution to the electrostatic free energy and obtain [formula: see text]. The number of protons released in the dissociation of the third strand from the target duplex at pH 7.0, delta n2, is thereby calculated to be 5.5, in good agreement with approximately six third strand dc residues per mole of triplex. This work shows that when third strand binding requires protonated residues that would otherwise be neutral, triplex formation and dissociation are mediated by proton uptake and release, i.e., a proton switch. As a by-product of this study, we have found that at low pH the Watson-Crick duplex d(A-G)6.d(C-T)6 undergoes a transition to a parallel Hoogsteen duplex d(A-G)6.d(C(+)-T)6.

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins

Background:Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF–DNA system has long been viewed as a paradigm of asymmetry in a protein–DNA interaction.Results We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting λ site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences.Conclusion Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein–DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.