Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The twisting potential of DNA has been determined directly by a method that measures the cyclization probability or j-factor of EcoRI restriction fragments as a function of DNA twist. The cyclization probability is proportional to Kc, the equilibrium constant for cyclization of the restriction fragment via its cohesive ends (Shore et al., 1981). Here we vary the twist of the DNA by making small internal additions to or deletions from a 242 bp EcoRI restriction fragment. A series of 12 DNA molecules has been studied, which range in length from 237 to 254 bp. The cyclization probability is measured from the rates of covalent closure by phage T4 DNA ligase of two systems: (1) a linear restriction fragment in equilibrium with its cyclized form and (2) half molecules (cut by a blunt-end endonuclease) in equilibrium with joined half molecules. The striking result is that, in this DNA size range, the j-factor depends strongly on the fractional twist: the difference between the total helical twist and the nearest integer. Thus j depends in an oscillatory manner on DNA length between 237 and 254 bp with a period of about 10 bp. These data give the free energy of DNA twisting as a function of twist. The curve of j versus DNA length can be fitted to a harmonic twisting potential with a torsional constant of C = 2.4 X 10(-19) erg cm. This value is in reasonable agreement with different estimates of C made by Barkley & Zimm (1979: C = 1.8 X 10(-19) to 4.1 X 10(-19) erg cm) and is somewhat larger than the value obtained resulting from the kinetics of DNA twisting measured by fluorescence depolarization of ethidium intercalated into DNA (C = 1.4 X 10(-19) erg cm; Millar et al., 1982; Thomas et al., 1980) or from spin label studies (Hurley et al., 1982). Our experiments provide a direct measurement of the torsional free energy and they show that the DNA twisting potential is symmetric. Our experiments also indicate that the DNA helix is continuous, or nearly so, in a nicked circle; presumably this happens because the DNA stacking interaction maintains the double helix in register across a single-strand nick. As a consequence, the twist of a singly nicked DNA circle is integral for small (approximately equal to 250 bp) planar DNA circles and there is a change in twist upon cyclization.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Energetics of DNA twisting. I. Relation between twist and cyclization probability. 631 55

To test the influence of pyrimidine methyl groups on DNA flexibility and helix repeat, two sets of 14 mixed sequence DNA molecules, spanning a range of lengths from 158 to 180 base pairs, were cyclized with T4 DNA ligase. The two sets differed only in that the Cyt-5 positions of all cytosines (80-90 cytosine residues per molecule) were fully methylated in the members of one set. Determination of the molar cyclization factors, persistence lengths, helix repeats, and torsional elastic constants revealed no significant differences between the two sets. These results imply that, at least for mixed sequence DNA, the biological consequences of cytosine methylation are likely to derive from either local structural distortions in the helix, which do not propagate as altered twist, or from direct protein-methyl cytosine interactions.
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PMID:Investigation of the influence of cytosine methylation on DNA flexibility. 781 73

We have used 1H NMR spectroscopy to determine the structural changes induced in the DNA oligomer d(5'-GCGTACGC-3')2 upon conversion of the 4'-hydroxy-methyl-4,5',8-trimethylpsoralen-DNA furan-side monoadduct (MAf) to the interstrand cross-link (XL). The MAf is a photochemical intermediate on the path to interstrand XL and has the psoralen intercalated into the helix. The local DNA structure is distorted in both adducts, but it returns to normal within three base pairs. The formation of XL requires displacement of the psoralen toward the initially unmodified strand, accompanied by a change in the hybridization of the thymine C-5 and C-6 carbons and a change in the local helix twist. The MAf is intercalated in the helix. There is no significant bend in the helix axis of either the MAf or XL. There are significant changes in the local helix dynamics upon photoadduct formation that may be recognized by cellular DNA repair enzyme systems. We hypothesize that the repair enzymes target lesions by detecting the conformational flexibility of the sugar-phosphate backbone induced by DNA-damaging agents.
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PMID:DNA structural reorganization upon conversion of a psoralen furan-side monoadduct to an interstrand cross-link: implications for DNA repair. 789 69

The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding. In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands. Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase. The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10 degrees . The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree.
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PMID:H1 binding unwinds DNA. Evidence from topological assays. 895 84

A feature of Ig hypermutation is the presence of hypermutable DNA sequences that are preferentially found in the V regions of Ig genes. Among these, RGYW/WRCY is the most pronounced motif (G:C is a mutable position; R=A/G, Y=C/T, and W=A/T). However, a molecular basis for the high mutability of RGYW was not known until recently. The discovery that activation-induced cytidine deaminase targets the DNA encoding V regions, has enabled the analysis of its targeting properties when expressed outside of the context of hypermutation. We analyzed these data and found evidence that activation-induced cytidine deaminase is the major source of the RGYW mutable motif, but with a new twist: DGYW/WRCH (G:C is the mutable position; D=A/G/T, H=T/C/A) is a better descriptor of the Ig mutation hotspot than RGYW/WRCY. We also found evidence that a DNA repair enzyme may play a role in modifying the sequence of hypermutation hotspots.
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PMID:Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. 1500 35

Smc2/4 forms the core of the Saccharomyces cerevisiae condensin, which promotes metaphase chromosome compaction. To understand how condensin manipulates DNA, we used two in vitro assays to study the role of SMC (structural maintenance of chromosome) proteins and ATP in reconfiguring the path of DNA. The first assay evaluated the topology of knots formed in the presence of topoisomerase II. Unexpectedly, both wild-type Smc2/4 and an ATPase mutant promoted (+) chiral knotting of nicked plasmids, revealing that ATP hydrolysis and the non-SMC condensins are not required to compact DNA chirally. The second assay measured Smc2/4-dependent changes in linking number (Lk). Smc2/4 did not induce (+) supercoiling, but instead induced broadening of topoisomer distributions in a cooperative manner without altering Lk(0). To explain chiral knotting in substrates devoid of chiral supercoiling, we propose that Smc2/4 directs chiral DNA compaction by constraining the duplex to retrace its own path. In this highly cooperative process, both (+) and (-) loops are sequestered (about one per kb), leaving net writhe and twist unchanged while broadening Lk. We have developed a quantitative theory to account for these results. Additionally, we have shown at higher molar stoichiometries that Smc2/4 prevents relaxation by topoisomerase I and nick closure by DNA ligase, indicating that Smc2/4 can saturate DNA. By electron microscopy of Smc2/4-DNA complexes, we observed primarily two protein-laden bound species: long flexible filaments and uniform rings or "doughnuts." Close packing of Smc2/4 on DNA explains the substrate protection we observed. Our results support the hypothesis that SMC proteins bind multiple DNA duplexes.
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PMID:The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. 1610 Jan 11

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.
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PMID:The effect of changes in the bulk dielectric constant on the DNA torsional properties. 1787 31

Aberrant methylation of promoter CpG islands is causally linked with a number of inherited syndromes and most sporadic cancers, and may provide valuable diagnostic and prognostic biomarkers. In this report, we describe an approach to simultaneous analysis of multiple CpG islands, where methylation-specific oligonucleotide probes are joined by ligation and subsequently amplified by polymerase chain reaction (PCR) when hybridized in juxtaposition on bisulfite-treated DNA. Specificity of the ligation reaction is achieved by (i) using probes containing CpGpCpG (for methylated sequences) or CpApCpA (for unmethylated sequences) at the 3' ends, (ii) including three or more probes for each target, and (iii) using a thermostable DNA ligase. The external probes carry universal tails to allow amplification of multiple ligation products using a common primer pair. As proof-of-principle applications, we established duplex assays to examine the FMR1 promoter in individuals with fragile-X syndrome and the SNRPN promoter in individuals with Prader-Willi syndrome or Angelman syndrome, and a multiplex assay to simultaneously detect hypermethylation of seven genes (ID4, APC, RASSF1A, CDH1, ESR1, HIN1 and TWIST1) in breast cancer cell lines and tissues. These data show that ligation of oligonucleotide probes hybridized to bisulfite-treated DNA is a simple and cost-effective approach to analysis of CpG methylation.
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PMID:A ligation assay for multiplex analysis of CpG methylation using bisulfite-treated DNA. 1799 53

The molecular structure of the DNA double helix has been known for 60 years, but we remain surprisingly ignorant of the balance of forces that determine its mechanical properties. The DNA double helix is among the stiffest of all biopolymers, but neither theory nor experiment has provided a coherent understanding of the relative roles of attractive base stacking forces and repulsive electrostatic forces creating this stiffness. To gain insight, we have created a family of double-helical DNA-like polymers where one of the four normal bases is replaced with various cationic, anionic or neutral analogs. We apply DNA ligase-catalyzed cyclization kinetics experiments to measure the bending and twisting flexibilities of these polymers under low salt conditions. Interestingly, we show that these modifications alter DNA bending stiffness by only 20%, but have much stronger (5-fold) effects on twist flexibility. We suggest that rather than modifying DNA stiffness through a mechanism easily interpretable as electrostatic, the more dominant effect of neutral and charged base modifications is their ability to drive transitions to helical conformations different from canonical B-form DNA.
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PMID:Mechanical properties of DNA-like polymers. 2401 60