Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases

Małgorzata Figiel¹, Marta Gapińska¹, Mariusz Czarnocki-Cieciura¹, Weronika Zajko¹, Małgorzata Sroka¹, Krzysztof Skowronek², Marcin Nowotny¹

¹Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland
²Biophysics and Structural Biology Facility, International Institute of Molecular and Cell Biology, Warsaw, Poland

Bacterial reverse transcriptases (RTs) can be classified into over 20 lineages, out of which only retrons, type II introns and diversity generating retroelements have been extensively characterized so far (1). Among RTs which are not yet well-understood, there is a distinct group of Abi polymerases. They are involved in abortive infection — antiviral defense strategy leading to suicide of the infected cell (2). Abi polymerases can be divided into three separate clads – AbiA, Abi-P2 and AbiK. They are unique in their ability to synthesize DNA products without either a template or a primer, utilizing hydroxyl groups of their own amino acids for priming instead (3).

In our study, we present structures of two Abi polymerases, belonging respectively to AbiK and Abi-P2 families. They form C-shaped, bilobal structures with RT-like domain on one side and a unique helical domain on the other. AbiK adopts a hexameric form, while Abi-P2 adopts a trimeric one, which is a feature unprecedented for RTs. These oligomeric states have been confirmed both by cryo-electron microscopy and MALS studies. Activity assays were performed to confirm priming role of an AbiK tyrosine covalently attached to DNA and to identify priming residue of Abi-P2. In summary, our data reveal a structural basis for a unique template independent, protein-primed polymerization mechanism in Abi RTs.

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2. J. T. Rostøl and L. Marraffini, “(Ph)ighting Phages: How Bacteria Resist Their Parasites,” Cell Host Microbe, vol. 25, no. 2, pp. 184–194, Feb. 2019.

3. C. Wang, M. Villion, C. Semper, C. Coros, S. Moineau, and S. Zimmerly, “A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro,” Nucleic Acids Res., vol. 39, no. 17, pp. 7620–7629, Sep. 2011