scholarly journals Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

ACS Nano ◽  
2016 ◽  
Vol 10 (12) ◽  
pp. 11127-11135 ◽  
Author(s):  
Cherie S. Tan ◽  
Jan Riedl ◽  
Aaron M. Fleming ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Bond Formation ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Kinetics Of

scholarly journals Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

2019 ◽  
Vol 47 (14) ◽  
pp. 7147-7162 ◽  
Author(s):  
Adele Williamson ◽  
Hanna-Kirsti S Leiros
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Bond Formation ◽  
Strand Break ◽  
Nucleophilic Attack ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

Kinetics of Coordinate Bond Formation. V. Kinetics of the Reaction P(CH3)3+BF3

1961 ◽  
Vol 34 (3) ◽  
pp. 715-717 ◽  
Author(s):  
G. B. Kistiakowsky ◽  
C. E. Klots
Keyword(s):  
Bond Formation ◽  
Coordinate Bond ◽  
Kinetics Of

scholarly journals In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

2020 ◽  
Vol 117 (11) ◽  
pp. 5741-5748 ◽  
Author(s):  
Travis Walton ◽  
Saurja DasGupta ◽  
Daniel Duzdevich ◽  
Seung Soo Oh ◽  
Jack W. Szostak
Keyword(s):  
Origin Of Life ◽  
In Vitro Selection ◽  
Random Sequence ◽  
Bond Formation ◽  
Rna Sequences ◽  
Phosphodiester Bond ◽  
Complex Protein ◽  
The Origin Of Life ◽  
Group 2

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide–activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

scholarly journals A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

2011 ◽  
Vol 16 (5) ◽  
pp. 486-493 ◽  
Author(s):  
Adam B. Shapiro ◽  
Ann E. Eakin ◽  
Grant K. Walkup ◽  
Olga Rivin
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Fluorescence Resonance

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

scholarly journals Kinetics of bond formation in cross-linked gelatin gels

2006 ◽  
Vol 125 (17) ◽  
pp. 174903 ◽  
Author(s):  
T. Abete ◽  
E. Del Gado ◽  
D. Hellio Serughetti ◽  
L. de Arcangelis ◽  
M. Djabourov ◽  
...  
Keyword(s):  
Bond Formation ◽  
Gelatin Gels ◽  
Kinetics Of
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