Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra 400076, India.
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra 400076, India.
Nucleic Acids Res. 2019 Jul 26;47(13):6932-6945. doi: 10.1093/nar/gkz236.
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.
多噬棘阿米巴原虫巨大病毒是一种感染阿米巴的巨型病毒，拥有超过 1000 个基因，其中包括几个参与 DNA 复制和修复的基因。在这里，我们报告了 mimivirus 基因产物 577（gp577）的生化特征，gp577 是一种假定蛋白（L537 基因的产物）。序列分析和系统发育表明，gp577 是一种引发酶-聚合酶（PrimPol）——这是在核质大 DNA 病毒（NCLDV）中首次鉴定的 PrimPol。纯化的重组 gp577 蛋白作为同源二聚体存在，并在环状和线性单链 DNA 模板上表现出从头 RNA 以及 DNA 合成。此外，gp577 使用脱氧核糖核苷酸（dNTPs）或核糖核苷酸（NTPs）延伸与 DNA 或 RNA 模板退火的 DNA/RNA 引物，证明其具有 DNA/RNA 聚合酶和逆转录酶活性。我们还表明，gp577 具有末端转移酶活性，能够使用 NTPs 和 dNTPs 延伸 ssDNA 和 dsDNA。gp577 的保守引发酶模体残基的突变导致引发酶、聚合酶、逆转录酶和末端转移酶活性的丧失。此外，我们表明 gp577 具有跨损伤合成（TLS）活性。Mimiviral gp577 代表第一个具有引发酶、聚合酶、逆转录酶、末端转移酶和 TLS 活性的 NCLDV 蛋白。
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