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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 tendency for relatively short (less than 500 base-pair) DNA molecules to circularize in the presence of DNA ligase is a sensitive function of both the lateral and torsional flexibilities of the molecules being studied. This tendency is reflected in a quantity known as the j-factor, which is determined experimentally by measuring the relative rates of circle and linear dimer formation at a specified concentration of linear monomer. Shimada & Yamakawa have provided an analytical representation of j that takes account of DNA molecules whose ends are not torsionally aligned. Their approach, however, assumes that contributions from helix writhe are small. Using a Monte Carlo approach for the determination of j, thereby avoiding any assumptions regarding writhe, we demonstrate that the computed, torsion angle-averaged quantity, [j], is exactly reproduced by the corresponding Shimada & Yamakawa quantity for all lengths examined. However, for DNA molecules having lengths that are substantially greater than the persistence length, P, the analysis of experimental ring-closure data using j (Shimada & Yamakawa) may lead to underestimates for the torsional elastic constant C. We demonstrate that no single set of values for P, C and the helical repeat (hR) can produce a reasonable fit of the computed j curve to the experimental values of Shore et al. This observation suggests that P, C and/or hR vary within the set of DNA molecules studied by those authors. The current computational analysis considers the effects on j of single or multiple bends in the helix axis. For single, centrally located bends, the shift in the distribution of end-to-end separations to smaller values is nearly offset by the less favorable polar alignment of the ends of the chain; the net effect being a modest change in j that is not a monotonic function of the bend angle. In contrast, polar alignment, and hence j, can be enhanced dramatically for molecules containing multiple, phased bends. However, for studies of the distribution of circle sizes formed from ligation of bend-containing DNA oligomers, the DNA lengths giving rise to maximal j values are smaller than predicted on the basis of the number of bends and the per-bend angle. This last result suggests that such studies may yield apparent bend angles that are too large.
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PMID:Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. I. Computational analysis. 231 3

We determined the magnitude of the bend induced in DNA by an adenine-thymine tract by measuring the rate of cyclization of DNA oligonucleotides containing phased A tracts. A series of linear multimers with 2-bp single-stranded ends, in which the (A.T)6 tracts are separated by CG2-3C sequences and are positioned 10 and 11 bp apart alternately, were prepared from 21 bp long synthetic duplexed deoxyoligonucleotides. The cyclization rates of the multimers (105-210 bp) and the bimolecular association rate of the 84 bp long multimer were measured in the presence of DNA ligase. From the rate constants of the cyclization and bimolecular association reactions, ring closure probabilities were obtained for the multimers. The systematically bent molecules were simulated by Monte Carlo methods, and the ring closure probabilities were calculated for a given set of junction bend angles. By comparing the calculated values of ring closure probabilities to experimental values and adjusting the junction bend angles to fit experimental values, the extent of bending at the junctions (or the extent of bending for an adenine tract) was determined. We conclude that an A6 tract bends the DNA helix by 17-21 degrees.
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PMID:Determination of the extent of DNA bending by an adenine-thymine tract. 236 Nov 40

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.
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PMID:High-mobility-group 1 protein mediates DNA bending as determined by ring closures. 841 24

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.
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PMID:The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure. 936 32

We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both intra-molecular and inter-molecular ligations. In this paper, we found that histone H1 differentially enhances ligation reaction of DNA double-strand breaks (DSB). Histone H1 stimulated exclusively inter-molecular ligation reaction of DSB with DNA ligase IIIbeta and IV, whereas HMGB1 enhanced mainly intra-molecular ligation reaction. Electron microscopy of direct DNA-protein interaction without chemical cross-linking visualized that HMGB1 bends and loops linear DNA to form compact DNA structure and that histone H1 is capable of assembling DNA in tandem arrangement with occasional branches. These results suggest that differences in the enhancement of DNA ligation reaction are due to those in alteration of DNA configuration induced by these two linker proteins. HMGB1 and histone H1 may function in non-homologous end-joining of DSB repair and V(D)J recombination in different manners.
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PMID:Nucleosome linker proteins HMGB1 and histone H1 differentially enhance DNA ligation reactions. 1189 Jul 3