DNA cruciform arms nucleate through a correlated but non-synchronous cooperative mechanism

Inverted repeat (IR) sequences in DNA can form non-canonical cruciform structures to relieve torsional stress. We use Monte Carlo simulations of a recently developed coarse-grained model of DNA to demonstrate that the nucleation of a cruciform can proceed through a cooperative mechanism. Firstly, a twist-induced denaturation bubble must diffuse so that its midpoint is near the centre of symmetry of the IR sequence. Secondly, bubble fluctuations must be large enough to allow one of the arms to form a small number of hairpin bonds. Once the first arm is partially formed, the second arm can rapidly grow to a similar size. Because bubbles can twist back on themselves, they need considerably fewer bases to resolve torsional stress than the final cruciform state does. The initially stabilised cruciform therefore continues to grow, which typically proceeds synchronously, reminiscent of the S-type mechanism of cruciform formation. By using umbrella sampling techniques we calculate, for different temperatures and superhelical densities, the free energy as a function of the number of bonds in each cruciform along the correlated but non-synchronous nucleation pathways we observed in direct simulations.Comment: 12 pages main paper + 11 pages supplementary dat

Unusually Long Palindromes Are Abundant in Mitochondrial Control Regions of Insects and Nematodes

BACKGROUND: Palindromes are known to be involved in a variety of biological processes. In the present investigation we carried out a comprehensive analysis of palindromes in the mitochondrial control regions (CRs) of several animal groups to study their frequency, distribution and architecture to gain insights into the origin of replication of mtDNA. METHODOLOGY/PRINCIPAL FINDINGS: Many species of Arthropoda, Nematoda, Mollusca and Annelida harbor palindromes and inverted repeats (IRs) in their CRs. Lower animals like cnidarians and higher animal groups like chordates are almost devoid of palindromes and IRs. The study revealed that palindrome occurrence is positively correlated with the AT content of CRs, and that IRs are likely to give rise to longer palindromes. CONCLUSIONS/SIGNIFICANCE: The present study attempts to explain possible reasons and gives in silico evidence for absence of palindromes and IRs from CR of vertebrate mtDNA and acquisition and retention of the same in insects. Study of CRs of different animal phyla uncovered unique architecture of this locus, be it high abundance of long palindromes and IRs in CRs of Insecta and Nematoda, or short IRs of 10–20 nucleotides with a spacer region of 12–14 bases in subphylum Chelicerata, or nearly complete of absence of any long palindromes and IRs in Vertebrata, Cnidaria and Echinodermata

A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs

Theoretical Analysis of Competing Conformational Transitions in Superhelical DNA

We develop a statistical mechanical model to analyze the competitive behavior of transitions to multiple alternate conformations in a negatively supercoiled DNA molecule of kilobase length and specified base sequence. Since DNA superhelicity topologically couples together the transition behaviors of all base pairs, a unified model is required to analyze all the transitions to which the DNA sequence is susceptible. Here we present a first model of this type. Our numerical approach generalizes the strategy of previously developed algorithms, which studied superhelical transitions to a single alternate conformation. We apply our multi-state model to study the competition between strand separation and B-Z transitions in superhelical DNA. We show this competition to be highly sensitive to temperature and to the imposed level of supercoiling. Comparison of our results with experimental data shows that, when the energetics appropriate to the experimental conditions are used, the competition between these two transitions is accurately captured by our algorithm. We analyze the superhelical competition between B-Z transitions and denaturation around the c-myc oncogene, where both transitions are known to occur when this gene is transcribing. We apply our model to explore the correlation between stress-induced transitions and transcriptional activity in various organisms. In higher eukaryotes we find a strong enhancement of Z-forming regions immediately 5′ to their transcription start sites (TSS), and a depletion of strand separating sites in a broad region around the TSS. The opposite patterns occur around transcript end locations. We also show that susceptibility to each type of transition is different in eukaryotes and prokaryotes. By analyzing a set of untranscribed pseudogenes we show that the Z-susceptibility just downstream of the TSS is not preserved, suggesting it may be under selection pressure

Specific deletion of DNA sequences between preselected bases.

Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation

Cleavage within an RNase III site can control mRNA stability and protein synthesis in vivo.

We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript. In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger. It is proposed that the mode of cleavage within the RNase III site determines mRNA stability. Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation. In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times. This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels. In contrast, processing at the lambda int site reduces Int synthesis. Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. The end result appears to be determined by the mode of cleavage within the RNase III site