The use of diaminopurine to investigate structural properties of nucleic acids and molecular recognition between ligands and DNA.

2,6-Diaminopurine (DAP) is an analogue of adenine which can be converted to nucleotides that serve as substrates for incorporation into nucleic acids by polymerases in place of (d)AMP. It pairs with thymidine (or uracil), engaging in three hydrogen bonds of the Watson-Crick type. The result of DAP incorporation is to add considerable stability to the double helix and to impart other structural features, such as an altered groove width and disruption of the normal spine of hydration. DNA containing DAP may or may not be recognized by restriction endonucleases; RNA containing DAP may not engage in normal splicing. The DAP.T pair affects the local flexibility of DNA and impedes the interaction with helix bending proteins. By providing a non-canonical hydrogen bond donor in the minor groove and/or blocking access to the floor of that groove it strongly affects interactions with small molecules such as antibiotics and anticancer drugs. Examples which illustrate altered recognition of nucleotide sequences in DAP-containing DNA are presented: changed sites of cutting by bleomycin, photocleavage by uranyl nitrate and footprinting with mithramycin. Using DNA in which both A-->DAP and G-->Inosine substitutions have been made it is possible to assess precisely the role of the purine 2-amino group in ligand-DNA recognition.     

Footprinting of echinomycin and actinomycin D on DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine.

In order to clarify the role of the purine 2-amino group in the recognition of DNA by small molecules we have examined the binding of actinomycin D and echinomycin to artificial DNA molecules asymmetrically substituted with inosine and/or 2,6-diaminopurine (DAP) in one of the complementary strands. These DNAs, prepared by a method based upon PCR, present various potential sites for antibiotic binding, including several containing only a single purine 2-amino group in different configurations. The results show unambiguously that the presence of two 2-amino groups is mandatory for binding of actinomycin D to double-stranded DNA. In the case of echinomycin only one purine 2-amino group is required for remarkably strong binding to the asymmetric TpDAP.TpA dinucleotide step, but the CpDAP.TpI step (which also contains only a single purine-2 amino group) does not afford a binding site. Evidently, removing a 2-amino group (G-->I substitution) is dominant over adding one (A-->DAP substitution). No sequences containing just a single guanine residue are acceptable. The possibility is raised that replacing guanosine with inosine may do more than remove a group endowed with hydrogen bonding capability and interfere with ligand binding in other ways. The new methodology developed to construct asymmetrically substituted DNA substrates for this work provides a novel strategy that should be generally applicable for studying ligand-DNA interactions, beyond the specific interest in drug binding to DNA, and may help to elucidate how proteins and oligonucleotides recognize their target sites.     

Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase.

The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on K m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).     

The influence of the exocyclic amino group characteristic of GC base pairs on molecular recognition of specific nucleotide sequences in DNA by Berenil and DAPI

The expedient of preparing homologous DNA samples substituted with inosine for guanosine residues, 2,6-diaminopurine (DAP) for adenine residues, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for the drugs berenil [1,3-bis(4-phenylamidinium) triazene] and DAPI (4′,6-diamidino-2-phenyl indole) on DNA. The selectivity of these two minor groove binders for AT-rich sequences is seen to be radically altered in the substituted DNA molecules. Neither berenil nor DAPI bind to DAP-substituted DNA where all purine residues bear a 2-amino group. By contrast, they bind to AT-rich, IC-rich and even mixed sequences of the inosine DNA where all purine residues lack the 2-amino group. With the inosine and DAP double substituted DNA, both berenil and DAPI bind preferentially to IC-rich clusters instead of their canonical tracts endowed with an extra 2-amino group through substitution with DAP. These results establish that the location of the purine 2-amino group represents a critical determinant for recognition of DNA nucleotide sequences by the two drugs. © 1997 John Wiley & Sons, Ltd.     

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.     

Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA.     

DNA conformational analysis in solution by uranyl mediated photocleavage.

Uranyl mediated photocleavage of double stranded DNA is proposed as a general probing for DNA helix conformation in terms of minor groove width/electronegative potential. Specifically, it is found that A/T-tracts known to constitute strong distamycin binding sites are preferentially photocleaved by uranyl in a way indicating strongest uranyl binding at the center of the minor groove of the AT-region. The A-tracts of kinetoplast DNA show the highest reactivity at the 3'-end of the tract--as opposed to cleavage by EDTA/Fell--in accordance with the minor groove being more narrow at this end. Finally, uranyl photocleavage of the internal control region (ICR) of the 5S-RNA gene yields a cleavage modulation pattern fully compatible with that obtained by DNase I which also--in a more complex way--senses DNA minor groove width.     

DNA recognition by DNase I

Bovine pancreatic DNase I shows a strong preference for double-stranded substrates and cleaves DNA with strongly varying cutting rates suggesting that the enzyme recognises sequence-dependent structural variations of the DNA double helix. The complicated cleavage pattern indicates that several local as well as global helix parameters influences the cutting frequency of DNase I at a given bond. The high resolution crystal structures of two DNase I-DNA complexes showed that the enzyme binds tightly in the minor groove, and to the sugar-phosphate backbones of both strands, and thereby induces a widening of the minor groove and a bending towards the major grooves. In agreement with biochemical data this suggests that flexibility and minor groove geometry are major parameters determining the cutting rate of DNase I. Experimental observations showing that the sequence environmental of a dinucleotide step strongly affects its cleavage efficiency can be rationalized by that fact that six base pair are in contact with the enzyme. Mutational analysis based on the structural results has identified critical residues for DNA binding and cleavage and has lead to a proposal for the catalytic mechanism.     

Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes

Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

Autosomal recessive phosphorylase kinase deficiency in liver, caused by mutations in the gene encoding the beta subunit (PHKB).

The association of autosomal recessive phosphorylase kinase deficiency in liver of a 3 1/2-year-old female child with mutations in the gene encoding the common part of the beta subunit of phosphorylase kinase is reported. The proband had a severe deficiency of phosphorylase kinase in liver, while the phosphorylase kinase activity in erythrocytes was only slightly diminished. She had no symptoms of muscle involvement. The complete coding sequences of the liver gamma subunit and of the beta subunit of phosphorylase kinase of the proband were analyzed for the presence of mutations, by either reverse-transcribed PCR or SSCP analysis. Three deviations from the normal sequence were found in the region encoding the common part of the beta subunit of phosphorylase kinase-namely, a 1827G-->A (W609X) transition, a 2309A-->G (Y770C) transition, and a deletion of nucleotides 2896-2911-whereas no mutations were detected in the sequence encoding the liver gamma subunit of phosphorylase kinase. The 1827G-->A mutation and the deletion both result in the formation of early stop codons. Investigation of DNA showed that the deletion is caused by a splice-acceptor site mutation (IVS30(-1),g-->t). Family analysis revealed that the 1827G-->A and IVS30(-1),g-->t substitutions are located on different parental chromosomes and that compound heterozygosity for these mutations segregates with the disease. The 2309A-->G mutation was detected in 2%-3% of the normal population. Thus, it is concluded that the deficiency of phosphorylase kinase in this proband is caused by compound heterozygosity for the 1827G-->A and the IVS30(-1),g-->t mutations and that the 2309A-->G mutation is a polymorphism. This implies that a defect in the sequence encoding the common part of the beta subunit of phosphorylase kinase may present as liver phosphorylase kinase deficiency.     

DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex.

The structure of a complex between DNase I and d(GCGATCGC)2 has been solved by molecular replacement and refined to an R-factor of 0.174 for all data between 6 and 2 A resolution. The nicked octamer duplexes have lost a dinucleotide from the 3' ends of one strand and are hydrogen-bonded across a 2-fold axis to form a quasi-continuous double helix of 14 base-pairs. DNase I is bound in the minor groove of the B-type DNA duplex forming contacts in and along both sides of the minor groove extending over a total of six base-pairs. As a consequence of binding of DNase I to the DNA-substrate the minor groove opens by about 3 A and the duplex bends towards the major groove by about 20 degrees. Apart from these more global distortions the bound duplex also shows significant deviations in local geometry. A major cause for the observed perturbations in the DNA conformation seems to be the stacking type interaction of a tyrosine ring (Y76) with a deoxyribose. In contrast, the enzyme structure is nearly unchanged compared to free DNase I (0.49 A root-mean-square deviations for main-chain atoms) thus providing a rigid framework to which the DNA substrate has to adapt on binding. These results confirm the hypothesis that groove width and stiffness are major factors determining the global sequence dependence of the enzyme's cutting rates. The nicked octamer present in the crystals did not allow us to draw detailed conclusions about the catalytic mechanism but confirmed the location of the active site near H134 on top of the central beta-sheets. A second cut of the DNA induced by diffusion of Mn2+ into the crystals may suggest the presence of a secondary active site in DNase I.     

Differences in the accessibility of methylated and unmethylated DNA to DNase I.

DNase I binds in the minor groove of DNA and is used as an enzymatic tool to investigate the interaction of proteins with DNA. Here we show that the major groove located 5-methyldeoxycytidine can enhance or inhibit the cleavage rates of DNA by DNase I. This effect may be caused in part by changes in DNA structure affecting the accessibility of the minor groove of DNA to DNase I.     

DNase I susceptibility of bent DNA and its alteration by ditercalinium and distamycin

The bending of kinetoplast DNA from Crithidia fasciculata is thought to be related to the periodic distribution of AA or TT cluster sequences. The sensitivity to DNase I of the two strands of this DNA was analyzed at nucleotide resolution by sequencing gel electrophoresis. The effect on the DNase I cleavage pattern of two drugs, ditercalinium and distamycin, that are able to remove bending was analyzed. The same analysis was done on a pBR 322 DNA fragment of random sequence as a control. The periodic distribution of the AA or TT clusters in the bent DNA fragment was first analyzed by computing the autocorrelation function of the AA or TT clusters in the bent DNA fragment. It is shown that the AT tracts are on average 10.5 base pairs apart. This value is almost identical with that of the B-DNA helix pitch in solution [10.5 (Wang, 1979); 10.6 +/- 0.1 (Rhodes & Klug, 1980)]. To reveal the periodic pattern of DNase I cleavage on this bent DNA, alone or in presence of drugs, the cross correlation between the different bands obtained from DNAse I cleavage and the presence of AA or TT sequences was computed. This shows that GC and mixed sequences are the most sensitive regions. These data also suggest that there is a periodic fluctuation in the width of the minor groove in the bent fragment. Ditercalinium and distamycin alter the DNase I cutting pattern of the bent DNA fragment but in an inverse fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

103 Probing DNA shape and methylation state on a genomic scale with DNase I

DNA binding proteins find their cognate sequences within genomic DNA through recognition of specific chemical and structural features. Here, we demonstrate that high-resolution DNase I cleavage profiles can provide detailed information about the shape and chemical modification status of genomic DNA. Analyzing millions of DNA-backbone hydrolysis events on naked genomic DNA, we show that the intrinsic rate of cleavage by DNase I closely tracks the width of the minor groove. Integration of these DNase I cleavage data with bisulfite sequencing data for the same cell type genome reveals that the cleavage directly adjacent to CpG dinucleotides is enhanced at least eight-fold by cytosine methylation. This phenomenon we show is attributable to methylation-induced narrowing of the minor groove. Furthermore, we demonstrate that it enables simultaneous mapping of DNase I hypersensitivity and regional DNA methylation levels using dense in vivo cleavage data. Taken together, our results suggest a general mechanism through which CpG methylation can modulate protein–DNA interaction strength via the remodeling of DNA shape.     

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.     

Different conformational families of pyrimidine.purine.pyrimidine triple helices depending on backbone composition.

Different helical conformations of DNA (D), RNA (R), and DNA.RNA (DR) hybrid double and triple helices have been detected using affinity cleavage analysis. Synthetic methods were developed to attach EDTA.Fe to a single nucleotide on RNA as well as DNA oligonucleotides. Cleavage patterns generated by a localized diffusible oxidant in the major groove on the pyrimidine strand of four purine.pyrimidine double helices consisting of all DNA, all RNA, and the corresponding hybrids reveal that the relative cleavage intensity shifts to the 5' end of the purine strand increasingly in the order: DD < DR < RD < RR. These results are consistent with models derived from structural studies. In six pyrimidine.purine.pyrimidine triple helices, the altered cleavage patterns of the Watson-Crick pyrimidine strands reveal at least two conformational families: (i) D + DD, R + DD, D + DR, and R + DR and (ii) R + RD and R + RR.     

DNA structure influences sequence specific cleavage by bleomycin.

We have examined the cleavage of several synthetic DNA sequences by iron(II)-bleomycin. We find that, although bleomycin cuts mixed sequence DNAs with a preference for GC = GT > GA >> GG, it efficiently cleaves regions of (AT)n cutting exclusively at ApT, not TpA. Isolated ApT steps show very little cleavage while blocks of three or more contiguous ATs are cut as efficiently as GpT. This cleavage is specific for (AT)n, since sequences of the type (TAA)n.(TTA)n and (ATT)n.(AAT)n are hardly cut at all. No cleavage is observed at ApC or CpA within sequences of the type (AC)n.(GT)n; regions of An.Tn are also not cut. Although the cobalt-bleomycin complex (which binds to but does not cleave DNA) yields good DNase I footprints at GT and GC sites, no footprints are observed within (AT)n, suggesting that although the cleavage reaction is efficient, the binding affinity is relatively weak. We propose a model in which bleomycin cleavage is determined by local DNA structure, while strong binding requires the presence of a guanine residue.