Four Parallel Pathways in T4 Ligase‐Catalyzed Repair of Nicked DNA with Diverse Bending Angles

Abstract The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase–AMP complex and the ligase–AMP–DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase–AMP complex that persist in the ligase–AMP–DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.


Supporting tables
Table S1.DNA sequences used.

Figure S2 .
Figure S2.Typical FRET values for fluorescently labeled DNA.(A) A typical FRET trace of free DNA, with a FRET value of approximately 0.27.(B) Histogram of all high states FRET efficiency.It can be fitted with three Gaussian distributions, with the central FRET values, positions of the peaks, being 0.42, 0.67, and 0.88, respectively.

Figure S3 .
Figure S3.Conformational transitions of nonsealable nicked DNA in a ligase-AMP-DNA complex.(A) Schematic plot of the assay.Fluorescently labeled nicked DNA is immobilized on a glass surface, followed by the addition of T4 DNA ligase.Changes in fluorescence intensity are recorded in two channels, and the FRET value is calculated.The 2,3-dideoxyribonucleotide (ddC) block the final step of the nick-sealing reaction.(B) Four typical time traces of the conformational transitions.

Figure S4 .
Figure S4.Conformational transitions of other sealable nicked DNA during ligation.The DNA sequence at both sides of the nick were changed in relative to the Figure 2 were tested.They also show four typical time traces of the conformational transitions.

Figure S5 .
Figure S5.A typical FRET trace of dsDNA, which only shows a FRET value of approximately 0.27.

Figure S6 .
Figure S6.PAGE gel image revealed nicking endonuclease can be used to check whether T4 DNA Ligase repairs nick in DNA.(A) Schematic of cleavage product release assay for sealable nicked DNA (I) and ddC nicked DNA (II).(B) PAGE gel assay which shows the nicking endonuclease can split DNA which was not repaired by ligase.

Figure S7 .
Figure S7.Reconstruction of four ligase-AMP-DNA complexes by Negative-staining electron microscope.(A) Survey views of ligase-AMP-DNA complex (in red ellipses), ligase only (in cyan circle), and DNA only (cyan arrow) are highlighted, respectively.Scale bar, 20 nm.(B) Several typical ligase-AMP-DNA complexes extracted from the raw images were presented.(C) Typical 2D reference-free class averages of the four conformations, including one straight and three bent DNA conformations.(D) 3D density maps of the four complex conformations.The maps were Gaussian low-pass filtered to 10Å.(E) The refined structural coordinates were docked into the density maps of the four ligase-AMP-DNA complex density maps.T4 DNA ligases were colored in blue for OBD, red for DBD, and green for NTase.DNAs were colored in magenta.AMPs were colored in orange.

Figure S9 .
Figure S9.Four conformations of T4 DNA ligase with AMP revealed by Negative-staining TEM.(A) Survey view of T4 DNA ligase-AMP complex.Several ligases-AMP complexes are highlighted in cyan circles.Scale bar, 20 nm.(B-C) Typical 2D reference-free class averages (B), and three-dimensional density maps (C) of the four different conformations of T4 DNA ligase-AMP complex (denoted as Ligase-AMP Complex I, II, III, and IV).The maps were Gaussian low-pass filtered to 10Å.(D), The refined structural coordinates were docked into the density maps of the four conformations.T4 DNA ligases were colored in blue for OBD, red for DBD, and green for NTase.AMPs were colored in orange.

Figure S11 .
Figure S11.Time trace of backbone-atom root mean square deviations (RMSD) in the molecular dynamics simulation for the models of ligase-AMP-DNA complex conformations (A) and ligase-AMP complex conformations (B).

Figure S12 .
Figure S12.The preparation and verification of the DNA bow-like constructures.(A) Schematic of the synthesis process for circular ssDNA.After annealing and ligation, linear-linear ssDNA ligation, correctly circularized products, and unreacted linear ssDNA may be present.We separated the samples using 10% denaturing PAGE (with 8M urea) and isolated the DNA through ethanol precipitation.Partial digestion of linear DNA by EXO I before electrophoresis separation can be performed (optional).(B) Denaturing PAGE images(stained with GelRed) of the 80 nt ssDNA (30 nt linker) ligation products.(C) Denaturing PAGE images (stained with GelRed) of all circular DNA samples purified by electrophoresis separation and ethanol precipitation.The final purified circular ssDNA was determined to be >95% pure based on grayscale analysis.(D) Typical examination of circular ssDNA samples.Linear ssDNA is completely digested by EXO I, while circular ssDNA is resistant to digestion.The results show the examination of circular ssDNA samples with 30 nt and 40 nt linkers.Similar results were conducted for all circular samples.(E) Image of the annealed products of bow-like DNA examined using 10% PAGE (Cy3 channel).

Table S2 .
Summary of each simulation and corresponding MD time scale. )All simulations include SPC/E water box, which extends at least 10.0 Å from any solute atom. a