Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.

Autor:	Pimentel SC; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA., Upton HE; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA., Collins K; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA. Electronic address: kcollins@berkeley.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2022 Mar; Vol. 298 (3), pp. 101624. Date of Electronic Publication: 2022 Jan 21.
DOI:	10.1016/j.jbc.2022.101624
Abstrakt:	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. Competing Interests: Conflict of interest BoMoC variants with improved properties are included in patent applications filed by University of California, Berkeley, with S. C. P., H. E. U. and K. C. as named inventors. H. E. U. and K. C. are the founders of Karnateq Inc., which licensed the RT technology. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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