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Reactions were quenched by a vol:vol addition of ligase reaction quench at time points as indicated in each figure legend. The ligated product was analyzed by capillary electrophoresis as described previously. The method of initial rates was utilized to determine the Michaelis-Menten parameters k cat and K m. The rates were plotted against their respective substrate concentrations and fit with the Michaelis-Menten equation Eq 1 to extrapolate the kinetic parameters: 1 where K m is the Michaelis constant, [S] is the substrate concentration, [E] is the enzyme concentration, V 0 is the initial rate and k cat is the turnover number.
In the event of observed inhibition by higher concentrations of substrate, the data was fit by either a substrate inhibition model Eq 2 : 2 where K i is the substrate inhibition constant, [ 31 ] or a competitive substrate inhibition model for an ordered Bi-Bi Ping-Pong reaction: 3 where k cat is the turnover number, K m is the Michaelis constant for substrate binding K mATP is the Michaelis constant for ATP binding, and K i is the inhibitor binding constant [ 32 ].
All nonlinear least squares data fitting was performed with the KaleidaGraph software Synergy Software, Version 4. All reactions were performed a minimum of three times; reported initial rates are the average of the individual experiments, and error is reported as one standard deviation. K m is the Michaelis constant for the substrate and K i is the inhibition constant.
A K d for the inhibitor is calculated by multiplying the K i value by the number of nonspecific binding sites on the inhibitor N , calculated with Eq 5 : 5 where L is the total length of the oligonucleotide, l is the estimated DNA-binding footprint size for the ligase. This calculation assumes that the ligase would bind with equal affinity to all non-specific binding sites. The dialysis buffer was changed a minimum of 3 times, however the initial buffer change was limited to a 1-hour incubation to prevent precipitation of magnesium pyrophosphate; all subsequent buffer changes were performed for a minimum of 4 hours.
We observed 1. Maximal enzyme adenylylation was determined by the amount of 32 P detected after a second reaction time point. Reaction progress is reported as the fraction of this maximal adenylylation. Self-adenylylation single turnover rate was determined under saturating ATP conditions by fitting the data with a single exponential equation Eq 6 : 6 where A is the reaction amplitude, and k is the observed single turnover rate. Reported experimental data are the average of a minimum of three replicates, and error reported is the standard error of the measurements.
The substrate was assembled as indicated above. Reactions were performed in ligase-binding buffer 50 mM Tris pH 7. Initial studies to determine the kinetic parameters for nick sealing by a variety of ligases T4, PBCV-1 and Tth showed a significant reduction in initial velocity at high concentrations of ds-nDNA substrate, indicative of a previously unobserved substrate inhibition effect in these enzymes.
Fig 2A , S1 Fig. The data were fit to a classic uncompetitive substrate inhibition model Eq 2 to allow estimation of the Michaelis constant K m for productive substrate binding and the K i for the inhibitory substrate interaction. In addition to uncompetitive substrate inhibition, the inhibition could also potentially be explained by a competitive model, with nicked substrate binding to deadenylylated ligase inhibiting the self-adenylylation reaction through blocking binding of ATP [ 6 , 37 ].
Thus, the T4 substrate inhibition data was also fit with a competitive substrate inhibition model for an ordered Bi-Bi Ping-Pong mechanism Eq 3. The initial rates were plotted against their respective substrate concentrations and fit by: A. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates. Taken together, these results suggested that the inhibition is proportional to the total amount of dsDNA in the reaction, and was not dependent on end binding.
As a control, two non-ligatable nicks, which would be expected to bind competitively in place of ligatable substrate, were tested for their inhibitory effect S3 Fig. Thus T4 DNA ligase appears to be inhibited by dsDNA independent of the concentration of ends, but the interaction with the dsDNA backbone is clearly weaker than binding to a non-ligatable nick substrate analogue.
Error reported is the standard deviation for the replicates. Electrophoretic mobility shift assays EMSAs were performed to examine the effect of increased non-substrate dsDNA concentrations on the ability of the ligase to bind its 75mer-ds-nDNA substrate. ATP-free reaction conditions and deadenylylated T4 DNA ligase were used to prevent ligation of the nicked substrates upon binding to the ligase. The substrate was first shown to be effectively bound by the ligase Fig 4 , lanes 2—6.
Even the lowest concentration of dsDNA used nM was able to effectively compete the ligase from its preferred ds-nDNA substrate, with higher concentrations effectively completely competing the ligase off the nicked substrate. Fig 4 , lanes 7— This result is consistent with competitive binding by the ligase to non-substrate DNA, a likely mechanism of the observed inhibition of nick ligation rates.
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 nM— nM. To test the competitive inhibition theory, a constant concentration of n-dsDNA nicked substrate was reacted with a range of concentrations of non-substrate IdsDNA and the effect on initial velocity measured.
In order to extract the inhibition constant of dsDNA on T4 DNA ligase, a competitive inhibitor fit was utilized, Eq 4 where the observed rate with inhibitor was normalized to the reaction rate without inhibitor Fig 5. The estimated binding footprint size used was based upon the binding size observed for other crystalized DNA ligases, as there is no available structure for T4 DNA ligase. The K m which was utilized for this calculation was derived from the substrate inhibition fit for Fig 2A.
The data fitwell to this competitive inhibition model, suggesting that non-substrate DNA inhibits largely through competitive binding with nicked substrate. Competitive inhibition fitting utilizing Eq 4. The affinity per base pair can also be calculated utilizing Eq 5. Inhibition of ligation could result solely from non-nicked dsDNA blocking binding of nicked substrate; however, ligation rate could also be influenced by blocking the ability of the ligase to bind or react ATP in the self-adenylylation reaction.
A single turnover assay was utilized to examine the rate of T4 DNA ligase self-adenylylation in the presence or absence of non-nicked, non-substrate IdsDNA. Thus, non-substrate dsDNA can inhibit single-turnover self-adenylylation as well as the turnover rate of nick ligation. The determined rates for self-adenylylation of an uninhibited reaction, 2.
The reactions were fit to a single exponential equation Eq 6 to determine the reaction rate. Show Active Center. Enzyme Reaction EC: 6. CHEBI: May also position the pyrophosphate leaving group. Download: Image , Marvin File. Kiong Ho. You can also search for this author in PubMed Google Scholar. Correspondence to C. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions.
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