Abstract
Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polβ-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD+-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a “Swiss army knife” enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5′-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD+-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional.
Highlights
DNA base lesions are the most common type of genomic damage and pose a challenge to genome stability
The Polβ-like core of the eukaryotic DNA polymerases λ, μ, terminal deoxynucleotidyl transferase (TdT) and yeast Pol IV, is fused to an N-terminal BRAC1 carboxy terminus (BRCT) domain that interacts with nonhomologous end joining-DNA bound factors to recruit these polymerases to double-strand breaks[23,24,25,26,27,28]
The complete genome sequence of the P. globosa virus revealed an ORF corresponding to gene 401, which would encode for a 1093 amino acid protein whose N-terminal 379 amino acids have homology with family X DNA polymerases (30% identity with human Pol β) (Fig. 1a); and whose C-terminal domain (475–1050) has homology with the NAD+-dependent DNA ligases[36] (Fig. 1b)
Summary
DNA base lesions are the most common type of genomic damage and pose a challenge to genome stability. X DNA polymerases (PolXs), including mammalian Pol β5 and Pol λ6, 7, bacterial PolXs8–10 as well as the African Swine Fever Virus (ASFV) PolX11, 12, are involved in the filling-in step of BER These polymerases are relatively small, monomeric proteins that catalyze the insertion of a few nucleotides and lack an intrinsic proofreading activity[13]. In Bacterial and Archaeal PolXs, the C-terminus of the Polβ-like core is fused to a polymerase and histidinol phosphatase (PHP) domain[29], which contains 3′-5′ exonuclease, 3′-phosphatase and 3′-phosphodiesterase activities, and endows the polymerase with the capacity to process damaged 3′ termini[10, 30,31,32] It has an AP-endonuclease activity, which enables the enzyme to process an AP site and restore the original nucleotide[9, 10]. Whereas PolX and ligase are encoded by two independent genes in the Marseilleviridae, Mimiviridae and Poxviridae families, which belong to the NCLDV group, PgV-16T and its close relative CeV, a virus infecting the unicellular marine phytoplankton Haptolina (formerly Chrysochromulina) ericina, represent the first examples where the DNA ligase gene is fused to the C-terminus of the PolX coding region[36, 39]
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