Characterization of a Novelcis-synandtrans-syn-IIPyrimidine Dimer Glycosylase/AP Lyase from a Eukaryotic Algal Virus,Paramecium bursaria chlorellaVirus-1

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1 , which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1 , Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria – Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

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Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: implications for biological function

Nucleic Acids Research ◽

10.1093/nar/gkg749 ◽

2003 ◽

Vol 31 (19) ◽

pp. 5560-5567 ◽

Cited By ~ 31

Author(s):

K. L. Meadows

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Function ◽

Ap Lyase

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Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1)

Biochemistry ◽

10.1021/acs.biochem.7b00060 ◽

2017 ◽

Vol 56 (13) ◽

pp. 1899-1910 ◽

Cited By ~ 9

Author(s):

Tina A. Müller ◽

Michael A. Tobar ◽

Madison N. Perian ◽

Robert P. Hausinger

Keyword(s):

Biochemical Characterization ◽

Ap Lyase

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Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic ArchaeonThermoplasma volcanium

Archaea ◽

10.1155/2016/8734894 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Miki Fujii ◽

Chieri Hata ◽

Munetada Ukita ◽

Chie Fukushima ◽

Chihiro Matsuura ◽

...

Keyword(s):

Protein Gene ◽

Excision Repair ◽

Dna Lesions ◽

Dna Glycosylase ◽

Oxygen Species ◽

Dna Glycosylases ◽

Optimal Activity ◽

Base Excision ◽

Ap Lyase

The oxidation of guanine (G) to 7,8-dihydro-8-oxoguanine (GO) forms one of the major DNA lesions generated by reactive oxygen species (ROS). The GO can be corrected by GO DNA glycosylases (Ogg), enzymes involved in base excision repair (BER). Unrepaired GO induces mismatched base pairing with adenine (A); as a result, the mismatch causes a point mutation, from G paired with cytosine (C) to thymine (T) paired with adenine (A), during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeonThermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315) shares significant sequence homology with the DNA glycosylases ofMethanocaldococcus jannaschii(MjaOgg) andSulfolobus solfataricus(SsoOgg). The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed inEscherichia coliand purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP) lyase activities on GO/N (where N is A, T, G, or C) mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG) can. These results indicate that TvoOgg-encodingTVG_RS00315is a member of the Ogg2 family ofT. volcanium.

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