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

Abstract

Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK–DNA covalent adduct visualized interactions between the 3′ end of DNA and the active site and covalent attachment of the 5′ end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase–Cas1 fusion protein

“…De novo initiation of cDNA synthesis at the CC of a 3′-tRNA-like structure was shown previously for a mitochondrial retroplasmid RT ( 49 ). However, the more diverse sequences of spacers acquired by Mm RT-Cas1/Cas2 in vivo led us to consider an alternate mechanism based on findings that bacterial AbiK and Abi-P2 RTs function in abortive phage infection (abi) by using nontemplated protein priming to synthesize long “random” sequence ssDNAs that contribute to altruistic cell death ( 18 , 20 ). Protein priming of cDNA synthesis using an OH group of a tyrosine, threonine, or serine residue resulting in covalent attachment of labeled nucleotides to the protein is a well-characterized mechanism for the initiation of cDNA synthesis by a number of viral and cellular RTs ( 50 – 52 ).…”

“…4A and 6A). AbiK and Abi-P2 RTs have distinctive structural features that prevent binding of a template but leave the RT active site accessible to dNTPs (20), All pathways start with Rt-cas1/cas2 terminal transferase activity adding short 3′-dnA tails (dn n ) to host cellular or pathogen RnA fragments resulting from cleavage by Rnases that leave either a 3′ Oh or a 3′ phosphate that can be removed enzymatically to leave a 3′ Oh. On the left are pathways in which Rt-cas1/cas2 uses different mechanism (de novo initiation, protein priming, or exogenous or synthesized short dnA oligomer primers) to synthesize cdnAs that remain annealed to the RnA-dn template in an RnA-dn/cdnA duplex.…”

“…In addition, some type III CRISPR systems have an associated reverse transcriptase (RT), either coexpressed with Cas1/Cas2 or present as an RT-Cas1 fusion protein, some of which also have an N-terminal Cas6 domain that processes guide RNAs for incorporation into effector complexes (12)(13)(14)(15)(16). Most CRISPR-associated RTs are closely related to the RTs encoded by mobile group II introns, prolific bacterial retrotransposons whose dissociated RTs have evolved in different bacteria to perform a variety of cellular functions (17)(18)(19)(20)(21)(22). Four RT-Cas1 proteins have been shown to site-specifically integrate RNA as well as DNA into CRISPR arrays in vivo (12,(23)(24)(25), but the mechanisms by which RNA protospacers are reverse-transcribed and integrated into CRISPR arrays have remained unclear.…”

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein

“…18,20 Protein priming resulting in covalent attachment of labeled nucleotides to an OH group of a tyrosine, threonine or serine is a well-characterized mechanism for the initiation of DNA synthesis by a number of viral and cellular RTs, but generally occurs at a fixed cDNA initiation site for those cased studied in detail. 20,50–52…”

“…12,14,65 Mobile group II intron-encoded RTs, the likely direct or once removed ancestors of CRISPRassociated RTs, have throughout evolution exhibited remarkable flexibility to modulate the biochemical activities of their RT active site and acquire additional domain to perform cellular functions. Bacterial examples include diversity generating retroelement RTs, which preferentially mis-incorporate specific dNTPs into coding regions to enable host-phage tropism switching; 66,67 retron RTs and abortive phage infection (abi) RTs, which using different mechanisms to synthesize ssDNAs thought to trigger phage defense mechanisms; 18,20 and group II intron-like 4 (G2L4) RTs, which evolved to function in double-strand break repair via microhomology-mediated end joining by optimizing by optimizing the strong strand annealing activity of group II intron RTs. 22 Other striking examples are the evolution of mobile group II introns or their close relatives into the eukaryotic RNA splicing apparatus, including the core spliceosomal protein PrP8, which evolved from a group II intron-like RT and promotes RNA splicing by interacting with snRNAs, which evolved from group II intron RNA domains, as well as closely related RTs encoded by LINE and other eukaryotic non-LTR retrotransposons, which use variations of the reverse transcription and DNA integration mechanisms adapted to eukaryotic genomes and nuclear-cytoplasmic compartmention.…”