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.

Figure 1.

Sequence analysis of gp577. (A) Maximum likelihood phylogenetic tree of family B DNA polymerase from NCLDVs. Arrow shows the probable acquisition of gp577 by Mimiviridae group I members. Lineages A, B and C of Mimiviridae group I are colored in red, black and pink, respectively. Mimiviridae group II is colored in green. Scale bar represents number of substitutions per site. (B) Multiple sequence alignment of gp577 with mimiviral gp577-like proteins and AEPs from eukaryotes and archaea. Motifs I, II, III and zinc finger motif are shown in red, green, blue and yellow colors, respectively. Proteins are designated by their protein IDs and the species name is separated by an underscore. Alignment obtained from MUSCLE was manually edited and numbers in parenthesis between the motifs indicate the number of amino acids. (C) Maximum likelihood phylogenetic tree of mimiviral gp577-like proteins and AEPs. Domain organization of the proteins depicting the position of primase motifs and Zinc finger is also shown. Scale bar represents number of substitutions per site. The length of proteins in domain organization is shown approximately to scale and motifs I-III and zinc finger motif are shown in blue and red, respectively. Colors used: dark blue, eukaryotes; green, archaea.

Figure 2.

Purified gp577 exhibits RNA/DNA primase activity. (A) SDS-polyacrylamide gel analysis of the purified gp577 and its mutants. (B) Demonstration of primase activity of gp577 by a coupled primase-pyrophosphatase assay. φX174 circular single-stranded virion DNA (2 nM) was incubated with 80 μM of each NTP, 5 mM MgCl₂, 0.1 unit pyrophosphatase and 500 nM gp577 at 30°C for 30 min. Inorganic phosphate released was quantified spectrophotometrically by the addition of malachite green reagent. (C) PicoGreen-based primase assay with linear and circular ssDNA. (D) Autoradiogram showing the de novo addition of NTPs on circular and linear templates by gp577. (E) Formation of DNA primers does not occur efficiently with Mg²⁺as the cofactor even at gp577 concentration of 2000 nM. RNA and DNA synthesis was monitored by incorporation of [α-³²P] ATP and [α-³²P] dATP, respectively by gp577. (F) DNA primase activity with varying concentrations of the cofactor MnCl₂, lane 1, no cofactor, lanes 2–6: 0.1, 0.5, 1, 2 and 5 mM MnCl₂, respectively. (G) De novo synthesis of DNA on linear and circular ssDNA. (H) Screening of gp577 mutants of putative motifs I, II and III for RNA (H) and DNA (I) primase activities. Primase assay was performed using 200 nM of gp577 or its mutants, D92A, D151A, H173A, D213A and D239A.

Figure 3.

gp577 possesses terminal transferase activity. (A) Extension of a ssDNA labeled at the 5′ end with [γ-³²P] ATP by 100 nM gp577 using dNTPs and NTPs. (B) Terminal transferase activities of gp577 with 5′ [γ-³²P] ATP labeled unblocked and blocked templates with NTPs and dNTPs. (C and D) Template independent extension of homo-oligomeric sequences by gp577 using dNTPs and NTPs. Reactions with homo-oligomers were carried out using 500 nM gp577, 1 mM Mn²⁺ at 30°C for 30 min. (E) Extension of the 3′ end of a blunt dsDNA by gp577. (F and G) Probing the terminal transferase activity of gp577 mutants while utilizing dNTPs and NTPs, respectively. About 500 nM gp577 or its mutants and poly(T) as the substrate were used for these reactions.

Figure 4.

gp577 exhibits DNA/RNA polymerase activity. (A) Extension of the annealed DNA primer by NTPs and dNTPs. (B) Comparison of the RNA and DNA polymerase activities of gp577 and XT-20 commercial polymerase. (C and D) Complete extension of primer using dNTPs and NTPs, respectively in the presence of 1 mM Mn²⁺ as the cofactor and varying concentrations of gp577 (Lane 1: No enzyme control, lanes 2–6: 50, 100, 200, 500 and 1000 nM of purified gp577, respectively). The reactions were incubated at 30°C for 5 min. (E and F) DNA and RNA polymerase assay with gp577 mutants. About 100 nM of the wild-type or mutant enzymes were used for the assay. Mutants D92A, H173A and D213A showed either complete loss or reduction in polymerase activity.

Figure 5.

Effect of cofactors on gp577 processivity and fidelity. (A) Schematic showing the experimental setup for single nucleotide incorporation assay. (B and C) Incorporation of varying concentrations of individual NTPs or dNTPs against the templating dA was studied using 5 mM Mg²⁺ as the cofactor. (D and E) Extension of the 15-mer primer with varying concentrations of NTPs or dNTPs using 1 mM Mn²⁺ as the cofactor. Reactions were performed with 100 nM gp577 enzyme and incubated at 30°C for 5 min.

Figure 6.

gp577 demonstrates relaxed substrate specificity. (A) DNA template-dependent extension of a RNA primer with dNTPs and NTPs mediated by gp577. (B) gp577 incorporates dNTPs as well as NTPs while extending a DNA primer annealed to a RNA template. (C) Extension of a RNA primer in a RNA template-dependent manner by gp577 using dNTPs and NTPs. All assays were performed using 100 nM of wild-type gp577 with 1 mM MnCl₂ and incubated at 30°C for 5 min. (D–F) Screening of gp577 mutants for their ability to extend the three different primer-template combinations using dNTPs and NTPs. About 100 nM of each mutant was used for this assay in the presence of 1 mM Mn²⁺ as the cofactor.

Figure 7.

Translesion synthesis activity of gp577. (A) A schematic representation of the standing start and running start assay templates used to assess the translesion synthesis ability of gp577. * indicates the modified template guanosine base. Running start polymerization by gp577 on unmodified (B), N²-Bn-dG modified (C) or 8-oxo-dG modified (D) templates. The reactions were incubated with 100 nM gp577 enzyme at 30°C for 5, 15 or 30 min. Single nucleotide incorporation against the modified template shown in Figure 7A II using the N²-Bn-dG-modified template, in the presence of 1 mM Mn²⁺ (E) or 8-oxo-dG-modified template, in the presence of 1 mM Mn²⁺ (F). All single nucleotide incorporation assays were performed with 100 nM gp577 enzyme at 30°C for 5 min.

Figure 8.

Biochemical activities of gp577 and their probable in vivo roles. gp577 can utilize different DNA substrates viz. ssDNA, a primer-template complex or a dsDNA. The enzyme displays varied activities such as DNA/RNA primase (A), DNA/RNA-polymerase (PrimPol), reverse transcriptase (B) and terminal transferase (C). Interestingly, gp577 prefers to utilize NTPs over dNTPs shown by longer product formation while using the former. (D) Probable mechanisms utilized by gp577 upon encountering a DNA lesion. Owing to its substrate flexibility, it may utilize either NTPs or dNTPs to directly bypass the lesion (left) thus acting as a translesion synthesis polymerase. Alternatively, gp577 could completely skip the modified base and reprime synthesis downstream of the lesion thereby ensuring continued replication (right). Template (substrate) DNA is shown in black; dark blue indicates either DNA or RNA; sky blue and orange colors indicate the newly synthesized DNA and RNA products respectively, whereas the pink triangle indicates the modified DNA base. The homo-dimeric enzyme is shown in yellow.