A short adaptive path from DNA to RNA polymerases

Abstract

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic "steric-gate" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C-, 2′-fluoro-, or 2′-azido-modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2′O-methyl-DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the K m for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.processivity | protein engineering | second gate R eplicative polymerases require extraordinary specificity in substrate selection, incorporation, and replication both to ensure fidelity and to exclude noncognate and/or damaged nucleotides from the genome. A particular threat to DNA genome integrity are ribonucleotide triphosphates (NTPs), which are present in the cell at concentrations up to 100-fold in excess of the cognate deoxyribonucleotide triphosphates (dNTPs) (1-3) yet differ from them only by the presence of a 2′-hydroxyl(-OH) group. Indeed, although DNA polymerases have evolved to exclude NTPs from their active sites, incorporation does occur to a detectable degree, with significant implications for genome stability and repair (2, 4). This issue may be even more acute for thermophilic organisms, because high temperatures further increase genome instability by accelerating the spontaneous degradation of RNA (5). Control of NTP incorporation by DNA polymerases is therefore a paradigmatic case of the link between polymerase substrate specificity and genome stability.DNA polymerases from all three domains of life are known to use a common strategy to prevent NTP incorporation into the nascent strand, by exerting stringent geometric control of the chemical nature of the 2′ position of the incoming nucleotide through a single active site residue, the "steric gate" (6). This strategy is so efficient that mutation of the steric gate alone (e.g., to an amino acid with a smaller side chain) can reduce discrimination against NTP incorporation by several orders of magnitude (6-13). Howev...

Non-Enzymatic Oligomerization of 3’, 5’ Cyclic AMP

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Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

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