Mimivirus encodes a multifunctional primase with DNA/RNA polymerase, terminal transferase and translesion synthesis activities

Abstract Acanthamoeba polyphaga mimivirus is an amoeba-infecting giant virus with over 1000 genes including several involved in DNA replication and repair. Here, we report the biochemical characterization of gene product 577 (gp577), a hypothetical protein (product of L537 gene) encoded by mimivirus. Sequence analysis and phylogeny suggested gp577 to be a primase-polymerase (PrimPol)—the first PrimPol to be identified in a nucleocytoplasmic large DNA virus (NCLDV). Recombinant gp577 protein purified as a homodimer and exhibited de novo RNA as well as DNA synthesis on circular and linear single-stranded DNA templates. Further, gp577 extends a DNA/RNA primer annealed to a DNA or RNA template using deoxyribonucleoties (dNTPs) or ribonucleotides (NTPs) demonstrating its DNA/RNA polymerase and reverse transcriptase activity. We also show that gp577 possesses terminal transferase activity and is capable of extending ssDNA and dsDNA with NTPs and dNTPs. Mutation of the conserved primase motif residues of gp577 resulted in the loss of primase, polymerase, reverse transcriptase and terminal transferase activities. Additionally, we show that gp577 possesses translesion synthesis (TLS) activity. Mimiviral gp577 represents the first protein from an NCLDV endowed with primase, polymerase, reverse transcriptase, terminal transferase and TLS activities.

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Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽

10.7554/elife.09591 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 120

Author(s):

Todd Blevins ◽

Ram Podicheti ◽

Vibhor Mishra ◽

Michelle Marasco ◽

Jing Wang ◽

...

Keyword(s):

Rna Polymerase ◽

Cytosine Methylation ◽

De Novo ◽

Transferase Activity ◽

Small Interfering Rnas ◽

Pol Iv ◽

Nuclear Rna ◽

Rna Polymerase Iv ◽

Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

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De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.74.2.851-863.2000 ◽

2000 ◽

Vol 74 (2) ◽

pp. 851-863 ◽

Cited By ~ 217

Author(s):

Guangxiang Luo ◽

Robert K. Hamatake ◽

Danielle M. Mathis ◽

Jason Racela ◽

Karen L. Rigat ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Oligonucleotide Primer ◽

Transferase Activity ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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Low-bias ncRNA Libraries using Ordered Two-Template Relay: Serial Template Jumping by a Modified Retroelement Reverse Transcriptase

10.1101/2021.04.30.442027 ◽

2021 ◽

Author(s):

Heather E. Upton ◽

Lucas Ferguson ◽

Morayma M. Temoche-Diaz ◽

Xiaoman Liu ◽

Sydney C. Pimentel ◽

...

Keyword(s):

Reverse Transcriptase ◽

Biochemical Characterization ◽

Recombinant Expression ◽

Group Ii Intron ◽

Terminal Deoxynucleotidyl Transferase ◽

Transferase Activity ◽

Rna Sequences ◽

Rna Sequence ◽

End To End ◽

Group Ii

AbstractNon-long terminal repeat (non-LTR) and group II intron retroelements encode reverse transcriptases (RTs) that copy the retroelement transcript directly into host cell DNA, often at specific target sites. Biochemical characterization of these enzymes has been limited by recombinant expression and purification challenges, hampering understanding of their transposition mechanism and their exploitation for research and biotechnology. Properties of retroelement RTs substantiate their application for end-to-end RNA sequence capture. To investigate this utility, we first compared a non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT showed processive template jumping, producing one cDNA from discontinuous templates each copied end-to-end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3’ ends of single-stranded RNA or DNA. Combining these two types of activity with additional insights about non-templated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for linking Next Generation Sequencing (NGS) adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production using modified non-LTR retroelment RT for Ordered Two-Template Relay (OTTR) outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles (EVs), identifying miRNAs as well as myriad other non-coding (nc) RNAs and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.SignificanceRetrotransposons are non-infectious mobile genetic elements that proliferate in host genomes via an RNA intermediate that is copied into DNA by a reverse transcriptase (RT) enzyme. RTs are important for biotechnological applications involving information capture from RNA, since RNA is first converted into complementary DNA for detection or sequencing. Here, we biochemically characterize RTs from two retroelements and uncover several activities that allowed us to design a streamlined, efficient workflow for determining the inventory of RNA sequences in processed RNA pools. The unique properties of non-retroviral RT activities obviate many technical issues associated with current methods of RNA sequence analysis, with wide applications in research, biotechnology, and diagnostics.

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Directed Evolution of DNA Polymerase, RNA Polymerase and Reverse Transcriptase Activity in a Single Polypeptide

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.06.050 ◽

2006 ◽

Vol 361 (3) ◽

pp. 537-550 ◽

Cited By ~ 67

Author(s):

Jennifer L. Ong ◽

David Loakes ◽

Szymon Jaroslawski ◽

Kathleen Too ◽

Philipp Holliger

Keyword(s):

Rna Polymerase ◽

Reverse Transcriptase ◽

Directed Evolution ◽

Dna Polymerase ◽

Reverse Transcriptase Activity ◽

Transcriptase Activity ◽

Single Polypeptide

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De NovoRNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.01021-15 ◽

2016 ◽

Vol 36 (8) ◽

pp. 1248-1259 ◽

Cited By ~ 12

Author(s):

Yoshiko Maida ◽

Mami Yasukawa ◽

Kenkichi Masutomi

Keyword(s):

Rna Polymerase ◽

Reverse Transcriptase ◽

De Novo ◽

Telomerase Reverse Transcriptase ◽

Dependent Manner ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Human Carcinoma ◽

Content Type ◽

Secondary Sirnas

RNA-dependent RNA polymerase (RdRP) plays key roles in RNA silencing to generate double-stranded RNAs. In model organisms, such asCaenorhabditis elegansandNeurospora crassa, two types of small interfering RNAs (siRNAs), primary siRNAs and secondary siRNAs, are expressed; RdRP produces secondary siRNAsde novo, without using either Dicer or primers, while primary siRNAs are processed by Dicer. We reported that human telomerase reverse transcriptase (TERT) has RdRP activity and produces endogenous siRNAs in a Dicer-dependent manner. However,de novosynthesis of siRNAs by human TERT has not been elucidated. Here we show that the TERT RdRP generates short RNAs that are complementary to template RNAs and have 5′-triphosphorylated ends, which indicatesde novosynthesis of the RNAs. In addition, we confirmed short RNA synthesis by TERT in several human carcinoma cell lines and found that TERT protein levels are positively correlated with RdRP activity.

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Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.01462-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1858-1871 ◽

Cited By ~ 51

Author(s):

Stephen W. B. Fullerton ◽

Martina Blaschke ◽

Bruno Coutard ◽

Julia Gebhardt ◽

Alexander Gorbalenya ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Functional Characterization ◽

Transferase Activity ◽

Dependent Manner ◽

Rna Dependent Rna Polymerase ◽

Key Enzyme

ABSTRACT Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

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Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis

Journal of General Virology ◽

10.1099/vir.0.81802-0 ◽

2006 ◽

Vol 87 (9) ◽

pp. 2621-2630 ◽

Cited By ~ 26

Author(s):

Jacques Rohayem ◽

Katrin Jäger ◽

Ivonne Robel ◽

Ulrike Scheffler ◽

Achim Temme ◽

...

Keyword(s):

Time Course ◽

Rna Synthesis ◽

De Novo ◽

Polymerase Activity ◽

Opposite Polarity ◽

Transferase Activity ◽

Structural Protein ◽

Double Stranded Rna ◽

Terminal Transferase

Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

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