Monkeypox DNA Polymerase

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spinqualitylabelsall models testing Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction The structure represents the monkeypox virus (MPXV) DNA polymerase holoenzyme in a replicating state, resolved by cryo-EM at ~2.8 Å. It contains the catalytic polymerase F8 bound to its processivity cofactors A22 and E4, along with a primer–template DNA duplex and an incoming dTTP. The structure reveals a poxvirus-specific mechanism in which the A22–E4 heterodimer forms a forward sliding clamp that encircles the single-stranded template strand, enabling highly processive DNA synthesis. Biological Function F8 is the B-family DNA polymerase responsible for MPXV genome replication. A22 and E4 form a heterodimeric processivity factor that stabilizes the polymerase on DNA and promotes continuous elongation. Overall Architecture Stoichiometry: 1 F8 : 1 A22 : 1 E4 + primer–template DNA + incoming dTTP. F8 (polymerase): 1004 residues traced (last two residues missing); canonical B-family domains — NTD, 3′–5′ Exonuclease (Exo), palm, fingers, thumb — plus five poxvirus-specific insertions (largest named insert2). A22 (processivity factor): three domains — NTD, Middle (Mid), CTD. The Mid shows structural similarity to ligase adenylylation and OB-fold modules but lacks canonical ligase activity (putative active site is nonfunctional). E4 (uracil-DNA glycosylase homolog): ~218 residues, resembles VACV D4 and contacts F8 Exo directly. DNA binding & active site Bound nucleic acids: 22-nt template strand + 14-nt primer strand in the structure; incoming dTTP and a catalytic Mg²⁺ are observed. DNA path: duplex lies in groove between palm and thumb; single-stranded 5′ template exits through a channel formed by F8 NTD + Exo and E4, perpendicular to the duplex. Residues coordinating catalysis / substrate: conserved Asp residues D549 (motif A) and D753 (motif C) coordinate the catalytic metal; Y554 stacks the incoming ribose (steric gate against rNTPs); R634 and K661 stabilize triphosphate. These motifs closely mirror canonical B-family polymerases. Processivity mechanism — “forward sliding clamp” Key observation: A22–E4 folds back and, together with F8 NTD/Exo, forms a closed ring/channel that encircles the single-stranded template. Functional consequence: This encirclement prevents template dissociation and enables high processivity — a distinct forward sliding-clamp mode (encircling ss-template) versus PCNA-type backward clamps (encircling dsDNA) used in many other systems. Supporting biochemistry: Primer-extension assays show F8 alone is distributive (incorporates <14 nt), while addition/assembly with A22–E4 yields full-length extension (60 nt template) in a concentration-dependent manner; alanine scanning of E4 residues (e.g., W36, R39, N165) confirms residues critical for processivity. Notable structural features Fingers domain rotates (~17°) toward palm in replicating state — brings R634 / K661 closer to incoming dNTP triphosphate. Thumb wraps around duplex; DNA duplex remains B-form over the 14 base pairs modeled. A22 Mid domain resembles ligase O-adenylylation/OB modules but substitutions (hydrophobic/negative residues) likely prevent ATP binding — A22 is a degenerative ligase-like linker rather than an active ligase. 47 F8 residues directly contact DNA (29 to template strand, 18 to primer strand), mostly interacting with phosphodiester backbone rather than bases — explains sequence-independent elongation.

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Yana Fedotova, Eric Martz, Susana Retamal, Alicia Daeden, Wayne Decatur, Jaime Prilusky, Silky Srivastava, Eran Hodis