Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

Abstract

SignificanceDespite being evolved to process the four canonical nucleotides, DNA polymerases are known to incorporate and extend from modified nucleotides, which is the key to numerous core biotechnology applications. The structural basis for postincorporation elongation remained elusive. We successfully crystallized KlenTaq DNA polymerase in six complexes, providing high-resolution snapshots of the modification “moving” from the 3′ terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity. This highlights the unexpected plasticity of the system as origin of the broad substrate properties of the DNA polymerase and guide for the design of improved systems.

Engineering polymerases for applications in synthetic biology

“…Marx and coworkers investigated the substrate specificity of A- and B-family DNA polymerases by solving high resolution crystal structures of KlenTaq and Kod DNA polymerases bound to natural and base-modified substrates (Bergen et al ., 2012, 2013; Kropp et al ., 2018; Kropp et al ., 2019). The structures indicate that bulky modifications pass through a cavity that extends outside the enzyme active site.…”

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…Structures of polymerases in complex with modified substrates may therefore aid in both rational engineering and retrospective understanding of XNA polymerases. Recently, Kropp et al solved ternary structures of KlenTaq polymerase with a C5-modified cytidine at each of six successive positions [150] in the catalysis cycle, recapitulating the movement of the modified substrate through the polymerase's active site and revealing a surprising level of flexibility in both the modified nucleotide and the interacting polymerase residues to accommodate the modification.…”

Engineering-driven biological insights into DNA polymerase mechanism

“…Polymerase structures also have significant value in the engineering process, particularly from related structures harbouring a range of substrates, sampling mismatches [47,48], unnatural bases [49] and triphosphate analogues [50]. Those "families" of structures give a very detailed knowledge of spatial constraints that can affect polymerase function.…”