Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.

Conformation and rigidity of DNA microcircles containing waf1 response element for p53 regulatory protein.

The tumor-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely that these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences based on direct imaging of the topology of microcircles containing the sequences. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microcircles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are markedly puckered with a small excess of negatively writhed molecules. The average measured values of writhe are 0.109+/-0.013 (for 60 positively writhed molecules) and -0.098+/-0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in linking number for the positively and negatively writhed molecules prior to ligation, from which we derive a twist mismatch of 178 degrees (overtwist). This is 44.5 degrees for each 42-mer precursor containing a single waf1/cip1/p21 p53 response-element, in good agreement with the range of values deduced by indirect biochemical techniques. The two values of writhe may also be used to determine the ratio of the bending (B) to twisting (C) rigidity, yielding B/C=0.23. This is about one-third of the value for long, random-sequence DNA, suggesting that the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These results support the idea, proposed by us earlier, that torsional stress may play a role in the regulation of p53 binding through modulation of twist at the binding site.

Atomic force microscopy reveals kinks in the p53 response element DNA.

p53 is a 53 kDa nuclear phosphoprotein. Its function as a tumor suppressor critically lies in its ability to recognize its target DNA response elements as a tetramer. Here, we report the structural theme intrinsic to the response element DNA that governs this recognition phenomenon. The intrinsic flexibility or dynamic bending between two distinctly different, but naturally occurring p53 response elements has been compared by ring closure. Results show that DNA binding sites containing helically phased d(CATG.CATG) tetra-nucleotide sequences at the centers of quasi-dyad symmetry in each half-response site are more intrinsically flexible (i.e. preferentially bent under axial stress) than their d(CTTG.CTTG) counterparts. Intriguingly, p53 binding sites containing these more flexible d(CATG.CATG) sequence elements also exhibit a stronger tendency for tetrameric binding of the p53 DNA binding domain peptide. Examination of the shapes of DNA microcircles obtained by circularization of oligomers constructed from such flexible p53 target DNA sequences in tandem using MacMode atomic force microscopy directly revealed sequence-specific kinks in solution. The tetra-nucleotide sequence d(CATG.CATG) is highly conserved in most functional p53 response elements. Consequently, we propose that the sequence-specific kinks originating from d(CATG.CATG) sequences could be a common structural theme in p53 response elements and as evident from the results reported here, could be a determinant of binding site recognition by the p53 protein and the subsequent stability of the p53-DNA complex.

Intra- and interloop interactions in the folded G quartet structure of Oxytricha telomeric sequence.

The antiparallel intramolecular G quartet structure for the 3.5 copy Oxytricha telomeric sequence d(G4T4)3G4 has been established using a combination of spectroscopic and chemical probing methods. In the presence of Na+ ions, this sequence exhibits a circular dichroism spectrum with a positive band at 295 nm and a negative band around 265 nm, characteristic of an antiparallel G quartet structure. Further, we show that d(G4T4)3G4 adopts an antiparallel intramolecular G quartet structure even in K+ unlike d(G4T4G4). KMnO4 probing experiments indicated the existence of intra and interloop interactions in the Na+ induced structure. We have found that K+ not only increases the thermal stability of G quartet structure but also binds to the loop region and disrupts stacking and interloop interactions. Biological consequences of such cation-dependent conformational micro-heterogeneity in the loop region of G quartet structures is also discussed.

Structure and stability of human telomeric sequence.

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T2AG3)4, d(G3T2A)3G3, and d(G3T2AG3) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G3T2A)3G3 and d(T2AG3)4 assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G3T2AG3) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide d(G3T2AG3) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T2AG3)4 suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo.

To delineate the DNA structural elements responsible for transcriptional control in vivo, we have developed a novel approach taking advantage of the degeneracy of the genetic code. Using synthetic oligonucleotides as structural cassettes we have been able to replace, within a gene, segments of DNA coding for the same amino acid sequence but capable of adopting unusual DNA structures and monitor the effect of such structural elements on gene expression in vivo. We find that the presence of an inverted repeat sequence, with a potential to adopt cruciform structure, within the beta-galactosidase gene down regulates its expression in vivo.

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.

Simple repetitive sequences in the genome: structure and functional significance.

The current explosion of DNA sequence information has generated increasing evidence for the claim that noncoding repetitive DNA sequences present within and around different genes could play an important role in genetic control processes, although the precise role and mechanism by which these sequences function are poorly understood. Several of the simple repetitive sequences which occur in a large number of loci throughout the human and other eukaryotic genomes satisfy the sequence criteria for forming non-B DNA structures in vitro. We have summarized some of the features of three different types of simple repeats that highlight the importance of repetitive DNA in the control of gene expression and chromatin organization. (i) (TG/CA)n repeats are widespread and conserved in many loci. These sequences are associated with nucleosomes of varying linker length and may play a role in chromatin organization. These Z-potential sequences can help absorb superhelical stress during transcription and aid in recombination. (ii) Human telomeric repeat (TTAGGG)n adopts a novel quadruplex structure and exhibits unusual chromatin organization. This unusual structural motif could explain chromosome pairing and stability. (iii) Intragenic amplification of (CTG)n/(CAG)n trinucleotide repeat, which is now known to be associated with several genetic disorders, could down-regulate gene expression in vivo. The overall implications of these findings vis-à-vis repetitive sequences in the genome are summarized.

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.