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    A short adaptive path from DNA to RNA polymerases

    Cozens, C. and Pinheiro, Vitor B. and Vaisman, A. and Woodgate, R. and Holliger, P. (2012) A short adaptive path from DNA to RNA polymerases. Proceedings of the National Academy of Sciences of the United States of America 109 (21), pp. 8067-8072. ISSN 0027-8424.

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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 Km 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.


    Item Type: Article
    Keyword(s) / Subject(s): processivity, protein engineering, second gate
    School: Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences
    Research Centres and Institutes: Structural Molecular Biology, Institute of (ISMB)
    Depositing User: Sarah Hall
    Date Deposited: 04 Feb 2014 10:44
    Last Modified: 02 Aug 2023 17:09


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