Mapping the position of DNA polymerase-bound DNA templates in a nanopore at 5 A resolution.
DNA polymerases are molecular motors that catalyze template-dependent DNA replication, advancing along template DNA by one nucleotide with each catalytic cycle. Nanopore-based measurements have emerged as a single molecule technique for the study of these enzymes. Using the alpha-hemolysin nanopore, we determined the position of DNA templates bearing inserts of abasic (1',2'-dideoxy) residues, bound to the Klenow fragment of Escherichia coli DNA polymerase I (KF) or to bacteriophage T7 DNA polymerase. Hundreds of individual polymerase complexes were analyzed at 5 A precision within minutes. We generated a map of current amplitudes for DNA-KF-deoxynucleoside triphosphate (dNTP) ternary complexes, using a series of templates bearing blocks of three abasic residues that were displaced by approximately 5 A in the nanopore lumen. Plotted as a function of the distance of the abasic insert from n = 0 in the active site of the enzyme held atop the pore, this map has a single peak. The map is similar when the primer length, the DNA sequences flanking the abasic insert, and the DNA sequences in the vicinity of the KF active site are varied. Primer extension catalyzed by KF using a three abasic template in the presence of a mixture of dNTPs and 2',3'-dideoxynucleoside triphosphates resulted in a ladder of ternary complexes with discrete amplitudes that closely corresponded to this map. An ionic current map measured in the presence of 0.15 M KCl mirrored the map obtained with 0.3 M KCl, permitting experiments with a broader range of mesophilic DNA and RNA processing enzymes. We used the abasic templates to show that capture of complexes with the KF homologue, T7 DNA polymerase, yields an amplitude map nearly indistinguishable from the KF map.
Gyarfas
Brett
B
Department of Computer Engineering, Baskin School of Engineering, University of California, Santa Cruz, CA 95064, USA.
Olasagasti
Felix
F
Benner
Seico
S
Garalde
Daniel
D
Lieberman
Kate R
KR
Akeson
Mark
M
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D049329:Nanostructures; D013698:Templates, Genetic
D004247:DNA; D004259:DNA-Directed DNA Polymerase