Fig 1.
Original Proposed Ligation Reaction Model.
The proposed model for nick sealing by a DNA ligase follows three highly conserved nucleotidyl-transfer reactions. 1. ATP reacts with the ligase active site lysine generating a covalently bound a Lig-AMP enzyme form. 2. The AMP is transferred from the ligase active site lysine onto the 5’ PO4 of the nick. 3. AMP is released from the nick upon sealing of the nick by nucleophilic attack from the 3’ OH. Sealing is followed by the release of AMP and sealed dsDNA from the ligase, and then re-adenylylation of the enzyme for subsequent turnover.
Fig 2.
kcat/Km Curve for T4 DNA Ligase.
The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A. a classical uncompetitive substrate inhibition model (Eq 2), where kcat and Km Values of 0.44 s-1 ± 0.3 s-1 and 4 nM ± 1 nM respectively, were determined. The Ki value for substrate inhibition was calculated to be 590 nM ± 170 nM. B. A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism (Eq 3) kcat and Km values of 0.48 s-1 ± 0.3 s-1 and 4 nM ± 1 nM respectively, were determined. The Ki value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.
Fig 3.
Various Inhibitors Effects on Rate of Nick Sealing.
Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.
Fig 4.
Competition for ds-nDNA-Binding by dsDNA.
Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).
Fig 5.
Effect of Increasing DNA Concentration on ds-nDNA Sealing Rate.
Competitive inhibition fitting utilizing Eq 4. The Ki for the addition of a I-75-dsDNA substrate was determined to be 100 nM ± 20 nM. The affinity per base pair can also be calculated utilizing Eq 5. Utilizing a binding footprint size of 24 bp, the Ki is calculated as 10 μM ± 2 μM. All reactions were performed a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.
Fig 6.
Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate.
The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation (Eq 6) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s-1 ± 2 s-1. While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s-1 ± 0.5 s-1and the 10 μM inhibited reaction had a single turnover rate of 1.0 s-1 ± 1 s-1. All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.
Fig 7.
Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A. Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B. Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.