Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

DNA-sequence recognition by CAP: role of the adenine N6 atom of base pair 6 of the DNA site.

Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('Model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'Model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). One difference between the two models involves Glu181 of CAP. Model I predicts that Glu181 of CAP makes two specificity determining contacts: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site, and one H-bond with the adenine N6 atom of T:A at base pair 6 of the DNA half site. In contrast, Model II predicts that Glu181 makes only one specificity determining contact: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site. In the present work, we show that replacement of T:A at base pair 6 of the DNA half site by T:N6-methyl-adenine has no, or almost no, effect on the binding of CAP. We conclude, contrary to Model I, that Glu181 of CAP makes no contact with the adenine N6 atom of base pair 6 of the DNA half site.     

DNA stretching and extreme kinking in the nucleosome core

DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55° kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.     

1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element.

The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by H?rd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].     

Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro.

We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA

Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.     

Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants.

We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif. A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed. The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities. All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type. All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type. These results suggest the structural integrity of the mutant repressors. On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed. We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator. A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5. Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA. These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes.     

2'-deoxyribonolactone lesion in DNA: refined solution structure determined by nuclear magnetic resonance and molecular modeling

The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].     

Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d

Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (~60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant–DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (~50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (~80°). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower T_m values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA.     

Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from mammalian cells binds specifically to six pBR322 and M13mp8 DNA sequences but only when they are methylated at their CpG dinucleotide pairs. We cloned three high-affinity MDBP recognition sites from the human genome on the basis of their binding to MDBP. These showed much homology to the previously characterized prokaryotic sites. However, the human sites exhibited methylation-independent binding apparently because of the replacement of m5C residues with T residues. We also identified three other MDBP sites in the herpes simplex virus type 1 genome, two of which require in vitro CpG methylation for binding and are in the upstream regions of viral genes. A comparison of MDBP sites leads to the following partially symmetrical consensus sequence for MDBP recognition sites: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3'; m5Y (m5C or T), R (A or G), Y (C or T). This consensus sequence displays an unusually high degree of degeneracy. Also, interesting deviations from this consensus sequence, including a one base-pair deletion in the middle, are sometimes observed in high-affinity MDBP sites.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

A:T --> G:C base pair substitutions occur at a higher rate than other substitution events in Pms2 deficient mouse cells

The mismatch repair pathway involves multiple proteins that are required to correct DNA polymerase generated mismatches before they become mutations. It has been shown recently, that the predominant base-pair substitution events leading to loss of endogenous Aprt activity in Pms2 null mouse cells are A:T --> G:C mutations (Oncogene 21 (2002) 1768, Oncogene 21 (2002) 2840). To determine if this observation could be explained by an increased rate of A:T --> G:C mutations relative to other base-pair substitutions, we developed a reversion assay to examine G:C --> A:T, C:G --> A:T, and A:T --> G:C mutations within mouse Aprt in a Pms2 null mouse kidney cell line. The results demonstrated a 6-50-fold increase in the rate of the A:T --> G:C mutations relative to the other base-pair substitutions. Additional work demonstrated that growth of the Pms2 null cells in antioxidant containing medium reduced the rate of the A:T --> G:C mutations. The results are discussed with regards to the role of mismatch repair proteins in preventing base-pair substitutions, including those induced by oxidative stress.