Abstract
Broad evolutionary expansion of polymerase families has enabled specialization of their activities for distinct cellular roles. In addition to template-complementary synthesis, many polymerases extend their duplex products by nontemplated nucleotide addition (NTA). This activity is exploited for laboratory strategies of cloning and sequencing nucleic acids and could have important biological function, although the latter has been challenging to test without separation-of-function mutations. Several retroelement and retroviral reverse transcriptases (RTs) support NTA and also template jumping, by which the RT performs continuous complementary DNA (cDNA) synthesis using physically separate templates. Previous studies that aimed to dissect the relationship between NTA and template jumping leave open questions about structural requirements for each activity and their interdependence. Here, we characterize the structural requirements for cDNA synthesis, NTA, template jumping, and the unique terminal transferase activity of Bombyx mori R2 non-long terminal repeat retroelement RT. With sequence alignments and structure modeling to guide mutagenesis, we generated enzyme variants across motifs generally conserved or specific to RT subgroups. Enzyme variants had diverse NTA profiles not correlated with other changes in cDNA synthesis activity or template jumping. Using these enzyme variants and panels of activity assay conditions, we show that template jumping requires NTA. However, template jumping by NTA-deficient enzymes can be rescued using primer duplex with a specific length of 3′ overhang. Our findings clarify the relationship between NTA and template jumping as well as additional activities of non-long terminal repeat RTs, with implications for the specialization of RT biological functions and laboratory applications.
Highlights
Reverse transcriptases (RTs) are an evolutionarily diverse group of enzymes defined by their ability to synthesize DNA from an RNA template
B. mori R2 protein is a favorable system for characterization of the biochemical activities of a non-long terminal repeat (LTR) retroelement RT
The BoMoC variants described in this work tease apart RT active-site structural requirements for complementary DNA (cDNA) synthesis, nucleotide addition (NTA), and template jumping (Table 2), enabling future studies of how these RT activities support non-LTR retroelement mobility
Summary
Reverse transcriptases (RTs) are an evolutionarily diverse group of enzymes defined by their ability to synthesize DNA from an RNA template. A later-branching non-LTR retroelement group is distinguished by its typical encoding of two ORFs: an ORF1 RNA binding protein that in in some retroelement lineages has other roles as well, and an ORF2 protein with an N-terminal apurinic/apyrimidinic EN domain followed by an RT domain [4,5] This group is generally less specific for an insertion site. TPRT is an activity specific to non-LTR retroelement RTs, other RNA-templated polymerases including RdRPs, group II intron RTs, and retroviral RTs show some ability to template-jump under individually optimized reaction conditions in vitro [25,26,27,28,29]. Our findings provide insight into the requirements for cDNA synthesis initiation by the TPRT mechanism of non-LTR retroelement mobility and will enable expansion of RT applications to research and medicine
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