Abstract
Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.
Highlights
Replicative DNA polymerases are highly faithful enzymes responsible for maintaining the genomic integrity of an organism [1]
Crystal structures of thermostable DNA polymerases, like KlenTaq DNA polymerase (Klenow fragment of Thermus aquaticus DNA polymerase I, A-family) and the archaeal B-family polymerases of 9◦N and Kod (Thermococcus kodakarensis), reveal that these bulky modifications pass through a large cavity that extends outside the enzyme active site [14,15,16]
Analysis of the resulting primer-extension reactions by denaturing polyacrylamide gel electrophoresis (PAGE) demonstrates that natural Bst DNA polymerase is capable of catalyzing full-length DNA synthesis on natural (DNA), non-cognate (RNA), and synthetic congener (FANA and threofuranosyl nucleic acid (TNA)) templates
Summary
Replicative DNA polymerases are highly faithful enzymes responsible for maintaining the genomic integrity of an organism [1]. Static structures depicting the four key mechanistic steps of the replication cycle (translocation, substrate binding, pre-catalysis, and post-catalysis) and their associated conformational changes are well documented [9] Together, these structures expand our understanding of the mechanism of DNA synthesis by illuminating the reaction pathway. Crystal structures of thermostable DNA polymerases, like KlenTaq DNA polymerase (Klenow fragment of Thermus aquaticus DNA polymerase I, A-family) and the archaeal B-family polymerases of 9◦N and Kod (Thermococcus kodakarensis), reveal that these bulky modifications pass through a large cavity that extends outside the enzyme active site [14,15,16] This cavity enables A- and B-family polymerases to incorporate C5-modified pyrimidines and C7-modified purines into the growing DNA strand and to continue synthesis after phosphodiester bond formation is complete
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