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.

Coevolutionary and Phylogenetic Analysis of Mimiviral Replication Machinery Suggest the Cellular Origin of Mimiviruses

“…2006 ). For the analysis, we have not considered the standalone primases, gp577 and gp857 as biochemical characterization showed that mimiviral gp577 is a primase-polymerase (PrimPol) that is involved in repair rather than replication and gp857 appears to be a paralog of gp577 ( Gupta et al. 2019 ).…”

“…In addition to the core replication proteins, namely, DNA polymerase (gp351), origin binding protein (gp1), primase-helicase bifunctional proteins (gp229), SSBP (gp544), sliding clamps (gp886, gp532, gp124), clamp loaders (gp549, gp538, gp513, gp425, gp441), ligase (gp331), RNase H (gp326, gp371), and topoisomerases (gp243, gp515, gp216), Mimivirus encodes several primases (gp577, gp857), helicases (gp612, gp132, and gp635), and bifunctional primase-helicase (gp8) proteins. Although their role in genome replication is not clear, it was recently shown that some of them, such as gp577, gp857, and gp8, do not appear to be involved in DNA replication ( Gupta et al. 2019 ).…”

The deubiquitinase USP36 Regulates DNA replication stress and confers therapeutic resistance through PrimPol stabilization

“…PrimPol is an important component to maintain adaptive DNA replication by repriming replication restart downstream of replication stalling lesions and structures ( 38 , 39 ). Understanding how cells adapt to genotoxic agents and how PrimPol mediates responses in replication fork restart has become increasingly important to optimize tumor therapy.…”

Site‐Specific N2‐dG DNA Adducts: Formation, Synthesis, and TLS Polymerase‐Mediated Bypass

“…Primer extension studies were further conducted for N 2 ‐Bn‐dG with gp577, a viral PrimPol protein encoded by mimivirus employing different cofactors (magnesium and manganese metal ions), NTPs (nucleotide triphosphates) and dNTPs. [ 91 ] Bypass efficiency of gp577 was promoted by manganese (over magnesium), which compromises the fidelity of the enzyme across from N 2 ‐Bn‐dG adduct. [ 91 ]…”

“…[ 91 ] Bypass efficiency of gp577 was promoted by manganese (over magnesium), which compromises the fidelity of the enzyme across from N 2 ‐Bn‐dG adduct. [ 91 ]…”