Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.
Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase.
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DOI：
10.1016/j.jbc.2022.101624
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Collins K
中科院分区：
文献类型：
--
作者：
Pimentel SC;Upton HE;Collins K
聚合酶家族的广泛进化扩展使得它们的活性能够专门用于不同的细胞作用。除了模板互补合成之外，许多聚合酶通过非模板核苷酸加成（NTA）来延伸其双链体产物。这种活性被用于克隆和测序核酸的实验室策略，并且可能具有重要的生物学功能，尽管后者在没有功能分离突变的情况下进行测试具有挑战性。几种逆转录元件和逆转录病毒逆转录酶（RT）支持NTA和模板跳跃，RT通过使用物理上分离的模板进行连续的互补DNA（cDNA）合成。先前的研究旨在剖析NTA和模板跳跃之间的关系，留下了关于每种活动的结构要求及其相互依赖性的开放性问题。在这里，我们的特点的cDNA合成，NTA，模板跳跃，和独特的终端转移酶活性的家蚕R2非长末端重复逆转录酶的结构要求。序列比对和结构建模，以指导诱变，我们产生跨基序的酶变体一般保守或特定的RT亚组。酶变体具有不同的NTA谱，与cDNA合成活性或模板跳跃的其他变化无关。使用这些酶变体和活性测定条件的面板，我们表明模板跳跃需要NTA。然而，NTA缺陷酶的模板跳跃可以使用具有特定长度的3 ′突出端的引物双链体来拯救。我们的研究结果阐明了NTA和模板跳跃以及非长末端重复RT的额外活动之间的关系，对RT生物学功能的专业化和实验室应用具有重要意义。
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.
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DOI：
10.1126/science.aba0372
发表时间：
2020-08-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gao L;Altae-Tran H;Böhning F;Makarova KS;Segel M;Schmid-Burgk JL;Koob J;Wolf YI;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
4.8
作者：
Bibillo, A;Eickbush, TH
通讯作者：
Eickbush, TH
影响因子：
64.8
作者：
Ghanim GE;Fountain AJ;van Roon AM;Rangan R;Das R;Collins K;Nguyen THD
通讯作者：
Nguyen THD
DOI：
10.1073/pnas.081077198
发表时间：
2001-04-24
影响因子：
11.1
作者：
Kim, MJ;Kao, C
通讯作者：
Kao, C