Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease

Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.     

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.     

Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.     

Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase

A complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.     

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.     

Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease

Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups. Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol(-1) toward specificity, with an additional 6-10 kcal mol(-1) arising from indirect readout. Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA.     

Minor groove functional groups are critical for the B-form conformation of duplex DNA

Two analogue bases are described: 3-deazaadenine is a derivative of adenine from which N3 has been deleted and 3-methyl-2-pyridone is a C-nucleoside that mimics thymine but lacks the O2 carbonyl. The dc(3)A-dm(3)2P base pair is similar to dA-dT but eliminates the polar functional groups in the minor groove. The presence of this base pair in dA-dT rich sequences results in destabilized duplexes or conformational preferences for monomolecular hairpins rather than bimolecular duplexes. When present in dG-dC rich sequences, no significant differences in helix stability are observed. These differences are explained on the basis of hydration effects, most notably, the elimination of the minor groove spine of hydration normally present in dA-dT rich sequences. CD spectra suggest that sequences with a fully modified core (four analogue base pairs) are more A-like helices than B-like helices. Sequences containing two analogue base pairs can be transformed to A-like helices under conditions of high salt, or 65% trifluoroethanol. These conformational changes are also explained in terms of a loss of hydration in the minor groove that normally stabilizes the B-form conformation. In the absence of such hydration, the helices are conformationally mobile and adopt a more A-like helix form.     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.     

Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.     

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.     

Interaction Between the Guanine Amino Group and the Adenine Six Membered Ring Stabilizes the Unusual Conformation of the CpA Step in B-DNA

Abstract

The CpA step is dramatically overwound in several B-DNA oligonucleotide crystal structures and its AT pair is substantially shifted towards the cytosine of the preceding base pair and towards the minor groove. We show using a geometrical analysis of the crystal data and empirical potential calculations that a strong interaction between the guanine amino group and the adenine six membered ring is responsible for the unique conformational properties of the CpA step.     

Impact of base pair identity 5′ to the spliceosomal branch site adenosine on branch site conformation

The branch site helix from Saccharomyces cerevisiae with pseudouridine (ψ) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine_{U2 strand}-pyrimidine_{intron strand} (R_U2-Y_intron) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A_U2-U_intron to U_U2-A_intron resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R_U2-Y_intron orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U → G•A or U•C), was markedly quenched, implying that the residue was stacked in the helix. The A 5′ to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R_U2-Y_intron base pair orientation in the ψ-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.     

Demethylation of thymine residues affects DNA cleavage by endonucleases but not sequence recognition by drugs.

The 5-methyl group of thymidine residues protrudes into the major groove of double helical DNA. The structural influence of this exocyclic substituent has been examined using a PCR-made 160 bp fragment in which thymidine residues were replaced with uridine residues. We show that the dT-->dU substitution and the consequent deletion of the methyl group affects the cleavage of DNA by deoxyribonuclease I and micrococcal nuclease. Analysis of the DNase I cleavage sites, in terms of di and trinucleotides, indicates that homopolymeric tracts of d(AT) become significantly more susceptible to DNase I cleavage when uridine is substituted for thymidine residues. The results indicate that removal of the thymidine methyl groups from the major groove at AT tracts induces structural perturbations that transmit into the opposite minor groove, where they can be detected by endonuclease probing. In contrast, DNase I footprinting experiments with different mono and bis-intercalating drugs reveal that dT-->dU substitution does not markedly affect sequence-specific drug-DNA recognition in the minor or major groove of the double helix. The consequences of demethylation of thymidine residues are discussed in terms of changes in the minor groove width connected to variations in the flexibility of DNA and the intrinsic curvature associated with AT tracts. The study identifies the methyl group of thymine as an important molecular determinant controlling the width of the minor groove and/or the flexibility of the DNA.     

The structure of DAPI bound to DNA

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.     

Effects of the minor groove pyrimidine nucleobase functional groups on the stability of duplex DNA: the impact of uncompensated minor groove amino groups

DNA sequences containing four types of analog nucleosides are described. All four are pyridine derivatives constructed as C-nucleosides so that they mimic the pyrimidine derivatives 2'-deoxyuridine, thymidine or 2'-deoxycytidine, but in all cases the analogs lack the corresponding O2-carbonyls that in duplex DNA are located in the minor groove. In place of the O2-carbonyl is a hydrogen atom, a polar fluorine atom, or a nonpolar methyl group. The described C-nucleosides have native-like bidentate Watson-Crick hydrogen-bonding faces and can form essentially normal W-C base pairs of varying stability with A or G. In each modified base pair, two inter-residue hydrogen bonds should be present. In spite of a common number of interstrand hydrogen bonds, the thermodynamic stabilities of the prepared duplexes, each containing two analog base pairs, vary dramatically. Most notably, base pairs containing uncompensated purine amino groups (those lacking a hydrogen-bonding partner) in the minor groove exhibit the most dramatic reductions in thermodynamic stability. Removal of such uncompensated amino groups results in increased duplex stability. Base pairs containing fluorine in the minor groove positioned adjacent to an amino group seem to enhance duplex stability marginally (relative to --H or --CH(3)), but there is little evidence to suggest that fluorine is an effective hydrogen-bonding partner in these systems. The presence of minor groove methyl groups results in the least stable duplexes in each series of sequences.     

DNA containing the base analogue 2-aminoadenine: preparation, use as hybridization probes and cleavage by restriction endonucleases.

The base analogue 2-aminoadenine (2,6-diaminopurine, D) has been introduced at selected positions into synthetic oligodeoxyribonucleotides and DNA by the combined use of chemical and enzymatic methods. 2-aminoadenine substitution for adenine introduces changes in the minor groove of DNA and creates an additional hydrogen bond in the Watson-Crick base pair with thymine. Oligonucleotide hybridization probes containing 2-aminoadenine showed increased selectivity and hybridization strength during DNA-DNA hybridization to phage or genomic target DNA. Properties of the base analogue with respect to DNA modifying enzymes were examined. 2-aminoadenine was used to probe minor groove determinants during the treatment of DNA by 12 restriction endonucleases. Inhibition of cleavage was found for several restriction enzymes.     

Molecular recognition of B-DNA by Hoechst 33258.

The binding sites of Hoechst 33258, netropsin and distamycin on three DNA restriction fragments from plasmid pBR322 were compared by footprinting with methidiumpropyl-EDTA X Fe(II) [MPE X Fe(II)]. Hoechst, netropsin and distamycin share common binding sites that are five +/- one bp in size and rich in A X T DNA base pairs. The five base pair protection patterns for Hoechst may result from a central three base pair recognition site bound by two bisbenzimidazole NHs forming a bridge on the floor of the minor groove between adjacent adenine N3 and thymine O2 atoms on opposite helix strands. Hydrophobic interaction of the flanking phenol and N-methylpiperazine rings would afford a steric blockade of one additional base pair on each side.     

DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.