Characterization of four-stranded DNA and RNA fragments.

We have investigated the structures formed by deoxyribo- and ribooligonucleotides containing guanine-rich sequences. Inter- and intrastrand guanine tetrads are not only more stable in the presence of K+ than they are in the presence of Na+, but also more compact.     

Quadruplex DNA: sequence, topology and structure

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.     

Tetraplex folding of telomere sequences and the inclusion of adenine bases.

Telomeres are required for eukaryotic chromosome stability. They consist of regularly repeating guanine-rich sequences, with a single-stranded 3' terminus. Such sequences have been demonstrated to have the propensity to adopt four-stranded structures based on a tetrad of guanine bases. The formation of an intramolecular foldback tetraplex is associated with markedly increased mobility in polyacrylamide. Most telomeric sequences are based either on a repeat of d(TnGGGG) or d(TnAGGG) sequences. We have used a combination 7-deazaguanine or 7-deaza-adenine substitution, chemical modification and gel electrophoresis to address the following aspects of intramolecular tetraplex formation. (i) Intramolecular tetraplex formation by d(TTTTGGGG)4 sequences is prevented by very low levels of 7-deazaguanine substitution. This confirms the important role of guanine N7 in the formation of the tetraplex. (ii) The sequences d(TTAGGG)4 and d(TTTTAGGG)4 fold into tetraplexes. By contrast, the electrophoretic behaviour of d(TTTTGGGA)4, d(TTTTAGAG)4 and d(TTTTGAGA)4 does not indicate formation of stable intramolecular tetraplexes under available conditions. (iii) Selective 7-deazaguanine and 7-deaza-adenine substitutions in d(TTTTAGGG)4 give results consistent with tetraplex folding by the formation of three G4 tetrads, with the adenine bases formally part of the single-stranded loops, where they probably interact with thymine bases. These results demonstrate that eukaryotic cells appear to have selected just those sequences that can adopt the tetraplex conformation for their telomeres, while those that cannot have been avoided. This suggests that the conformation may be significant in the function of the telomere, such as attachment to nuclear structures.     

Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G-G-C sequence.

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.     

Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.

In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].     

In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance

Mammalian chromosomes terminate with a 3' tail which consists of reiterations of the G-rich repeat, d(TTAGGG). The telomeric tail is the primer for replication by telomerase, and it may also invade telomeric duplex DNA to form terminal lariat structures, or T loops. Here we show that the ubiquitous and highly conserved mammalian protein hnRNP D interacts specifically with the G-rich strand of the telomeric repeat. A single gene encodes multiple isoforms of hnRNP D. All isoforms bind comparably to the G-rich strand, and certain isoforms can also bind tightly and specifically to the C-rich telomeric strand. G-rich telomeric sequences readily form structures stabilized by G-G pairing, which can interfere with telomere replication by telomerase. We show that hnRNP D binding to the G-rich strand destabilizes intrastrand G-G pairing and that hnRNP D interacts specifically with telomerase in human cell extracts. This biochemical analysis suggest that hnRNP D could function in vivo to destabilize structures formed by telomeric G-rich tails and facilitate their extension by telomerase.     

Detection and quantification of adducts formed upon interaction of diamminedichloroplatinum (II) with DNA, by anion-exchange chromatography after enzymatic degradation

A method has been developed to determine the adducts formed upon interaction of cis- and trans-diamminedichloroplatinum(II) (cis- and trans-DDP) with DNA. After 5 h at 50 degrees C in the dark, the amount of cis-DDP bound to salmon sperm DNA was larger than the amount of the trans-isomer. After enzymatic degradation with deoxyribonucleases to nucleotides and Pt-containing (oligo)nucleotides, the various products were separated by DEAE chromatography and analyzed for Pt by flameless AAS. Indications were obtained for the presence of nucleotides containing monofunctionally bound Pt and of adducts originating from interstrand DNA crosslinks. DEAE chromatography of digests of cis-DDP-treated DNA yielded a product with overall charge -1, which was identified with NMR and CD as cis-[Pt(NH3)2-d(pGpG)], the oligonucleotide derived from intrastrand crosslinks between two adjacent guanines. Another major peak contained Pt-oligonucleotides with overall charge -2, which could be derived from intrastrand crosslinks between two guanines at sites with pGpXpG (X=T,C,A or G) base sequences.     

A Chlamydomonas protein that binds single-stranded G-strand telomere DNA.

We have identified a protein in Chlamydomonas reinhardtii cell extracts that specifically binds the single-stranded (ss) Chlamydomonas G-strand telomere sequence (TTTTAGGG)n. This protein, called G-strand binding protein (GBP), binds DNA with two or more ss TTTTAGGG repeats. A single polypeptide (M(r) 34 kDa) in Chlamydomonas extracts binds (TTTTAGGG)n, and a cDNA encoding this G-strand binding protein was identified by its expression of a G-strand binding activity. The cDNA (GBP1) sequence predicts a protein product (Gbp1p) that includes two domains with extensive homology to RNA recognition motifs (RRMs) and a region rich in glycine, alanine and arginine. Antibody raised against a peptide within Gbp1p reacted with both the 34 kDa polypeptide and bound G-strand DNA-protein complexes in gel retardation assays, indicating that GBP1 encodes GBP. Unlike vertebrate heteronuclear ribonucleoproteins, GBP does not bind the cognate telomere RNA sequence UUUUAGGG in gel retardation, North-Western or competition assays. Thus, GBP is a new type of candidate telomere binding protein that binds, in vitro, to ss G-strand telomere DNA, the primer for telomerase, and has domains that have homology to RNA binding domains in other proteins.     

The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands

Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH₃NH₂)₂Cl₂], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand cross-links. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH₃NH₂)₂Cl₂] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced frequency of intrastrand cross-links yielded by trans-[Pt(CH₃NH₂)₂Cl₂] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH₃NH₂)₂Cl₂]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.     

Reaction of nucleic acids with cis-diamminedichloroplatinum(II): interstrand cross-links

In the reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with double-helical (dC-dG)4.(dC-dG)4 or (dC-dG)5.(dC-dG)5, intrastrand and interstrand cross-links between two guanine residues are formed. This is shown by gel electrophoresis in denaturing conditions of the reaction products and by high-performance liquid chromatography (HPLC) analysis of the products digested with nuclease P1. In the reaction of cis-DDP and poly(dG-dC).poly(dG-dC), at relatively low levels of platination, it is mainly interstrand cross-links between two guanine residues that are formed. This is shown by HPLC analysis of the nuclease P1 digest and by gel electrophoresis in denaturing and nondenaturing conditions of the platinated polymer after cleavage with the restriction enzyme HhaI. Moreover, the antibodies to platinated poly(dG-dC).poly(dG-dC) cross-react with the interstrand cross-linked (dC-dG)4 or (dC-dG)5 but not with the intrastrand cross-linked (dC-dG)4 or (dC-dG)5. These antibodies cross-react with platinated natural DNA. The amount of interstrand cross-links deduced from radioimmunoassays (0.5% of the total bound platinum) is lower than that (2%) deduced by gel electrophoresis in denaturing conditions of a platinated DNA restriction fragment. By gel electrophoresis, it is also shown that in vitro the isomer trans-DDP is more efficient in forming interstrand cross-links than cis-DDP.     

A thymine tetrad in d(TGGGGT) quadruplexes stabilized with Tl+/Na+ ions

We report two new structures of the quadruplex d(TGGGGT)₄ obtained by single crystal X-ray diffraction. In one of them a thymine tetrad is found. Thus the yeast telomere sequences d(TG_1–3) might be able to form continuous quadruplex structures, involving both guanine and thymine tetrads. Our study also shows substantial differences in the arrangement of thymines when compared with previous studies. We find five different types of organization: (i) groove binding with hydrogen bonds to guanines from a neighbour quadruplex; (ii) partially ordered groove binding, without any hydrogen bond; (iii) stacked thymine triads, formed at the 3′ends of the quadruplexes; (iv) a thymine tetrad between two guanine tetrads. Thymines are stabilized in pairs by single hydrogen bonds. A central sodium ion interacts with two thymines and contributes to the tetrad structure. (v) Completely disordered thymines which do not show any clear location in the crystal. The tetrads are stabilized by either Na⁺ or Tl⁺ ions. We show that by using MAD methods, Tl⁺ can be unambiguously located and distinguished from Na⁺. We can thus determine the preference for either ion in each ionic site of the structure under the conditions used by us.     

Biophysical studies on the stability of DNA intrastrand cross-links of transplatin

Clinically ineffective transplatin [trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.     

Nuclear magnetic resonance solution structures of inter- and intrastrand adducts of DNA cross-linker SJG-136

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials for the treatment of malignant disease. Previous studies on the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, typified by SJG-136 and DSB-120 (2), have shown that these planar ligands react with the exocyclic NH(2) groups of two guanine bases in the base of the minor groove of DNA to form an irreversible interstrand cross-linked sequence-specific adduct. Using high-field NMR, we have characterized and modeled the previously predicted interstrand duplex adduct formed by SJG-136 with the self-complementary 5'-d(CICGATCICG)(2) duplex (4). This first SJG-136 NMR-refined adduct structure has been compared with previous high-field NMR studies of the adducts of the closely related PBD dimer DSB-120 with the same duplex and of the adduct of tomaymycin (3) formed with 5'-d(ATGCAT)(2). Surprisingly, the SJG-136 duplex adduct appears to be more closely related to the tomaymycin adduct than to the DSB-120 adduct with respect of the orientation and depth of insertion of the ligand within the minor groove. The intrastrand duplex adduct formed in the reaction of SJG-136 with the noncomplementary 5'-d(CTCATCAC)·(GTGATGAG) duplex (5) has also been synthesized and modeled. In this duplex adduct, the nature of the cross-link was confirmed, the central guanines were identified as the sites of alkylation, and the stereochemical configuration at C11 at both ends of the SJG-136 molecule was determined to be S. The NMR-refined solution structures produced for the intrastrand adduct confirm the previously proposed structure (which was based solely on mass spectroscopy). Both the inter- and intrastrand SJG-136 duplex adducts form with minimal distortion of the DNA duplex. These observations have an impact on the proposal for the mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and, potentially, on the type of pharmacodynamic assay to be used in clinical trials. SGJ-136 is currently in phase II clinical trials with several groups working on both dimeric cross-linking agents and monoalkylating ligands based on the PBD alkylating moiety. This study suggests subtle differences between the DNA binding of SJG-136 and the C2 unsubstituted analogue DSB-120 that are likely to be the origins of the differences in potency. Confirmation of the stereochemical configuration at the C11 position (particularly in the intrastrand adduct) provides confirmation of binding orientation that was previously only speculation in the HPLC MS study. Together, these observations are likely to be of value in the development of third-generation PBD-based cross-linkers and monoalkylating analogues.     

Formation of novel hairpin structures by telomeric C-strand oligonucleotides.

Telomeres are specialized structures at the ends of chromosomes that are required for long term chromosome stability and replication of the chromosomal terminus. Telomeric DNA consists of simple repetitive sequences with one strand G-rich relative to the other, C-rich, strand. Evolutionary conservation of this feature of telomeric repeat sequences suggests that they have specific structural characteristics involved in telomere function. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoretic analyses showed that telomeric C-strand oligonucleotides form stable non-Watson-Crick hairpin structures containing C.C+ base pairs. Formation of such hairpins may facilitate previously reported G-strand exclusive interactions.     

DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation,recognition by high mobility group domain proteins,and nucleotide excision repair

The novel phase II antitumor polynuclear platinum drug BBR3464 ([(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](NO(3))(4)) forms intra- and interstrand cross-links (CLs) on DNA (which is the pharmacological target of platinum drugs). We examined first in our recent work how various intrastrand CLs of BBR3464 affect the conformation of DNA and its recognition by cellular components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the present work, we have extended the studies on the DNA interstrand CLs of this drug. The results have revealed that the interstrand CLs are preferentially formed between guanine residues separated by 2 base pairs in both the 3' --> 3' and 5' --> 5' directions. The major 1,4-interstrand CLs distort DNA, inducing a directional bending of the helix axis and local unwinding of the duplex. Although such distortions represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of BBR3464. On the other hand, in contrast to intrastrand adducts of BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide excision repair. It has been suggested that interstrand CLs of BBR3464 could persist considerably longer in cells compared with intrastrand adducts, which would potentiate the toxicity of the interstrand lesions to tumors sensitive to this polynuclear drug.