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PubMed1990

Binding of the antitumor drug nogalamycin and its derivatives to DNA: structural comparison.

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-A resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6(1)) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences.

Base Composition↗

PubMed2004

Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin.

The parallel (recombination) 'R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T.A) and 6MI*(C.G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative 'branch migration' and 'strand exchange' structures and discuss why these are less likely than the R-type triplex.

2-Aminopurine↗

PubMed1998

Sequence-dependent DNA structure: the role of the sugar-phosphate backbone.

A detailed analysis of the coupling between the conformational properties of the sugar-phosphate backbone and the base stacking interactions in dinucleotide steps of double helical DNA is described. In X-ray crystal structures of oligonucleotides, the backbone shows one major degree of freedom, consisting of the torsion angles chi, delta, zeta and the pseudorotation phase angle, P. The remaining torsion angles (beta, epsilon, alpha and gamma) comprise two less important degrees of freedom. The base stacking interactions show three degrees of freedom: slide-roll-twist, shift-tilt, and rise (which is more or less constant). Coupling is observed between the base and backbone degrees of freedom. The major base stacking mode, slide-roll-twist, is coupled to the major backbone mode, chi-P-delta-zeta. The secondary base stacking mode, shift-tilt, is coupled to epsilon and zeta and to a lesser extent to the chi-P-delta-zeta mode. We show that the length of the backbone, C, given by the same strand C1'-C1' separation, is an excellent single parameter descriptor for the conformation of the backbone and the way in which it is coupled to the base stacking geometry. The slide-roll-twist motion relates to changes in the mean backbone length, C, and the shift-tilt motion to the difference between the lengths of the two backbone strands, DeltaC. We use this observation to develop a simple virtual bond model which describes the coupling of the backbone conformations and the base stacking geometry. A semi-flexible bond is used to connect the same strand C1'-C1' atoms. Analysis of the X-ray crystal structure database, simple geometric considerations and model building experiments all show that this bond is flexible with respect to slide, shift and propeller but rigid with respect to the other 14 local base stacking parameters. Using this simple model for the backbone in conjunction with potential energy calculations of the base stacking interactions, we show that it is possible to predict accurately the values of these 14 base step parameters, given values of slide, shift and propeller. We also show that the base step parameters fall into three distinct groups: roll, tilt and rise are determined solely by the base stacking interactions and are independent of the backbone; twist is insensitive to the base stacking interactions and is determined solely by the constraints of a relatively rigid fixed length backbone; slide and shift are the primary degrees of freedom and cannot be predicted accurately at the dinucleotide level because they are influenced by the conformations of neighbouring steps in a sequence. We have found that the context effect on slide is mediated by the chi torsion angles while the context effect on shift results from a BI to BII transition in the backbone. We have therefore reduced the dimensionality of the dinucleotide step problem to two parameters, slide and shift.

Carbohydrates↗

PubMed2001

Change of plasmid DNA structure, hypermethylation, and Lon-proteolysis as steps in a replicative cascade.

I have defined conditions under which RepFIC plasmid DNA can be maintained in a state of lowered helical density. In exponentially growing cultures, the DNA of lowered helical density is present in small amounts but never totally absent, suggesting that it is a normal variant of plasmid maintenance. It is fully methylated at frequent sites by dam-methyltransferase, some not previously recognized, further suggesting that the variant is a precursor of replication. The low-helical density plasmid is present in dam hosts, indicating that methylation is not essential for the change in helical density. The lowered helical density is stabilized in lon hosts, suggesting that Lon-protease may remove both the protein(s) that lower the helical density and the dam-methyltransferase after each round of replication.

ATP-Dependent Proteases↗

PubMed1983

DNA structural dynamics: longitudinal breathing as a possible mechanism for the B in equilibrium Z transition.

The transition from B-DNA to Z-DNA not only requires a change in the helix architecture from a right-handed to a left-handed form, but also requires that all of the base pairs be flipped over. A transition mechanism is proposed in which the base pairs flip over one at a time while keeping the Watson-Crick hydrogen bonds intact. The two base pairs on either side of the pair that turns over form a kind of dynamic sandwich around it, and the flip takes place in a cavity that is formed when the distance between them is increased from about 7A in the intact helix to about 14A in the transition state. Since the transition occurs in two steps (formation of the cavity, followed by the base pair flip) and since the cavity propagates down the helix after the base pair is turned over, the mechanism accounts for the cooperativity of the B in equilibrium Z transition.

Base Composition↗

PubMed2004

The role of DNA structure and dynamics in the recognition of bovine papillomavirus E2 protein target sequences.

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN(4)CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG.CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15 ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI <--> BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT.ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

Base Sequence↗

PubMed1988

Applications of the twist difference to DNA structural analysis.

The twist is a fundamental geometric property of nucleic acids. Calculation of the twist in the most general case requires detailed specification of the three-dimensional path of each strand, but many important cases may be analyzed by considering only the twist difference. If C1, C2, and C3 are three distinct space curves, the twist difference about C1 is defined as Tw(C3, C1) - Tw(C2, C1). We show here that this difference measures the rotation of the correspondence surface joining C1 to C2 about the correspondence surface joining C1 to C3. This result has application to DNA containing local nonuniformities, such as denatured regions, cruciforms, and other altered structures. It also facilitates the calculation of twist for three-stranded structures, including D-loops in mitochondrial DNA and replication and transcription intermediates. The twist difference may also be used to simplify greatly the analysis of twist changes in duplex DNA due to winding on surfaces, such as histones and certain enzymes. In such cases the strand-axis twist of DNA divides into two independent terms. The first term arises from the twist of the local reference frame, and the second arises from the rotation of either strand about the duplex axis as measured in the local reference frame. Twist changes consequent to nucleosome winding, for example, arise from the twist of the nucleosome axis, a straight line, about the DNA axis plus the rotation of either strand of the DNA about its axis in the reference frame of the cylinder.

DNA, Superhelical↗

PubMed2000

Sequence-dependent DNA structure: dinucleotide conformational maps.

We have used a computational model to calculate the potential energy surface for dinucleotide steps in double helical DNA as a function of the two principal degrees of freedom, slide and shift. By using a virtual bond to model the constraints imposed by the sugar-phosphate backbone, twist, roll, tilt and rise can be simultaneously optimised for any given values of slide and shift. Thus we have been able to construct complete conformational maps for all step types. For some steps, the maps agree well with experimental data from X-ray crystal structures, but other steps appear to be strongly perturbed by the effects of context (conformational coupling with the neighbouring steps). The optimised values of twist and roll show sequence-dependent variations consistent with the crystal structure data. The conformational maps allow us to construct adiabatic paths, and hence calculate the flexibility of each step with respect to slide and shift. Again the results agree well with the available experimental assignments of flexibility: YR steps, CA/TG and CG, are the most flexible and RR steps, such as AA, the least flexible.

Base Pairing↗

PubMed2001

Effects of DNA structure and homology length on vaccinia virus recombination.

Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting approximately 4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3'-to-5' exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.

Animals↗

PubMed1988

Slipped DNA structures within the enhancer region of the Moloney murine leukemia virus.

We have examined the S1 nuclease sensitivity of supercoiled plasmids harboring the Moloney Murine Leukemia Virus (MoMuLV) long terminal repeat (LTR). S1 sensitivity was found within the LTR enhancer direct repeats. Transformation of E. coli DH5 cells with a construct containing most of the MoMuLV LTR yielded the precise deletion of one direct repeat and loss of S1 sensitivity. The dependence of S1 sensitivity on the presence of both direct repeats, together with the exact excision of one direct repeat by E. coli, suggests the presence of slipped DNA within the enhancer. Such structures may represent targets for effector proteins which mediate vital functions during viral propagation.

Base Sequence↗

PubMed1988