Directed Evolution of DNA Polymerase, RNA Polymerase and Reverse Transcriptase Activity in a Single Polypeptide

Ong, Jennifer L.; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

doi:10.1016/j.jmb.2006.06.050

Cited by 89 publications

(98 citation statements)

References 37 publications

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“…The existence of a second checkpoint for RNA synthesis located in the thumb subdomain had been predicted by us and others (6,10,16,17) on the basis of the phenotype of steric-gate mutants and their premature termination of RNA synthesis, typically at n + 6/7. Indeed, eight of the nine mutations in the starting D4 polymerase (TgoT: L403P, P657T, E658Q, K659H, Y663H, E664Q, D669A, K671N, and T676I) with weak RNA polymerase activity are located in a sequence segment (P657-T676) in the center of the thumb subdomain and in close contact with the nascent strand around n + 6 (Fig.…”

Section: Discussionmentioning

confidence: 89%

“…The second gate may therefore be a specific adaptation to prevent the deleterious consequences of NTP misincorporation or replication of genomic lesions at high temperature (32). Conversely, despite significant structural variability in the elaboration of the thumb subdomain, extension checkpoints have been observed in different polymerase families, including polA, where similar n + 6 termination of RNA synthesis is observed in steric-gate mutants (16,17). In analogy to the diverse nature of steric-gate residues, the second gate may therefore be elaborated in different ways in the context of different thumb domain structures.…”

Section: Discussionmentioning

confidence: 99%

“…However, although steric-gate mutations generally render DNA polymerases permissive for NTP incorporation, they do not by themselves enable synthesis of RNAs beyond short termination products. Indeed, engineering efforts using rational design (9), in vitro and in vivo screening (14,15), or directed evolution by phage display (16) and compartmentalized self-replication (17) have so far only yielded polymerases that can synthesize RNAs up to 58 nucleotide incorporations (nt) long (14) and more commonly stall at +6-7 nt (14-16, 18) even after prolonged incubation. At the same time, there is compelling structural and phylogenetic evidence for an adaptive path linking DNA to RNA polymerase activity in the evolution of the single-subunit RNA polymerases (ssRNAPs) of mitochondria and T-odd bacteriophages (e.g., T7 RNA polymerase), which are thought to derive from an ancestral polA-family DNA polymerase (19)(20)(21)(22).…”

mentioning

confidence: 99%

“…At the same time, there is compelling structural and phylogenetic evidence for an adaptive path linking DNA to RNA polymerase activity in the evolution of the single-subunit RNA polymerases (ssRNAPs) of mitochondria and T-odd bacteriophages (e.g., T7 RNA polymerase), which are thought to derive from an ancestral polA-family DNA polymerase (19)(20)(21)(22). Thus, we (17) and others (6,10,16) have argued that there must be a determinant of polymerase substrate specificity that has remained unidentified and that precludes synthesis of longer RNAs in the steric-gate mutants.…”

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confidence: 99%

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A short adaptive path from DNA to RNA polymerases

Cozens

Pinheiro

Vaisman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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115

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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...

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A short adaptive path from DNA to RNA polymerases

Cozens

Pinheiro

Vaisman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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115

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“…Recent studies have shown that substrate specificity in four of these twelve overlapping classes can be modulated with a small number of mutations. For instance, it has been reported recently that a RNA polymerase function was obtained from a DNA polymerase using in vitro compartmentalization, and a mutant with a single mutation was among the optimal mutants at synthesizing RNA [20]. Also, in the case of a transporting ATPase, the specificity of transport from H + to Li + was achieved by just four mutations [21].…”

Section: Resultsmentioning

confidence: 99%

CORRIE: enzyme sequence annotation with confidence estimates

et al. 2007

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Using a previously developed automated method for enzyme annotation, we report the reannotation of the ENZYME database and the analysis of local error rates per class. In control experiments, we demonstrate that the method is able to correctly re-annotate 91% of all Enzyme Classification (EC) classes with high coverage (755 out of 827). Only 44 enzyme classes are found to contain false positives, while the remaining 28 enzyme classes are not represented. We also show cases where the re-annotation procedure results in partial overlaps for those few enzyme classes where a certain inconsistency might appear between homologous proteins, mostly due to function specificity. Our results allow the interactive exploration of the EC hierarchy for known enzyme families as well as putative enzyme sequences that may need to be classified within the EC hierarchy. These aspects of our framework have been incorporated into a web-server, called CORRIE, which stands for Correspondence Indicator Estimation and allows the interactive prediction of a functional class for putative enzymes from sequence alone, supported by probabilistic measures in the context of the pre-calculated Correspondence Indicators of known enzymes with the functional classes of the EC hierarchy. The CORRIE server is available at: http:/ /www.genomes.org/services/corrie/.

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In Vitro Compartmentalization (IVC) and Other High‐Throughput Screens of Enzyme Libraries

Aharoni

Tawfik

2008

Protein Engineering Handbook

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Directed Evolution of DNA Polymerase, RNA Polymerase and Reverse Transcriptase Activity in a Single Polypeptide

Cited by 89 publications

References 37 publications

A short adaptive path from DNA to RNA polymerases

A short adaptive path from DNA to RNA polymerases

CORRIE: enzyme sequence annotation with confidence estimates

In Vitro Compartmentalization (IVC) and Other High‐Throughput Screens of Enzyme Libraries

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