T4 DNA ligase is more than an effective trap of cyclized dsDNA.
T4 DNA ligase is used in standard cyclization assays to trap double-stranded DNA (dsDNA) in low-probability, cyclic or highly bent conformations. The cyclization probability, deduced from the relative yield of cyclized product, can be used in conjunction with statistical mechanical models to extract the bending stiffness of dsDNA. By inserting the base analog 2-aminopurine (2-AP) at designated positions in 89 bp and 94 bp dsDNA fragments, we find that T4 DNA ligase can have a previously unknown effect. Specifically, we observe that addition of T4 ligase to dsDNA in proportions comparable to what is used in the cyclization assay leads to a significant increase in fluorescence from 2-AP. This effect is believed to originate from stabilization of local base-pair opening by formation of transient DNA-ligase complexes. Non-specific binding of T4 ligase to dsDNA is also confirmed using fluorescence correlation spectroscopy (FCS) experiments, which reveal a systematic reduction of dsDNA diffusivity in the presence of ligase. ATP competes with regular DNA for non-covalent binding to the T4 ligase and is found to significantly reduce DNA-ligase complexation. For short dsDNA fragments, however, the population of DNA-ligase complexes at typical ATP concentrations used in DNA cyclization studies is determined to be large enough to dominate the cyclization reaction.
School of Chemical and Biomolecular Engineering, and Department of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Chongli
C
Yuan
Xiong Wen
XW
Lou
Elizabeth
E
Rhoades
Huimin
H
Chen
Lynden A
LA
Archer
D015075:2-Aminopurine / Q000032:analysis; D020029:Base Pairing; D003500:Cyclization; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry*; D004058:Diffusion; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence
D004270:DNA, Circular; D015075:2-Aminopurine; D004247:DNA; D011088:DNA Ligases