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  : 1  : 1 + – DNA + incoming .

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- F8) and D753 (motif C- A22) coordinate the catalytic metal; Y554 of F8 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.

The following figure shows two binding modes of processivity factors with polymerases. The processivity factors bound with template in poxvirus function as a “forward sliding clamp” (i) or dsDNA products in eukaryotes as a “backward sliding clamp” (ii).

Significant 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’s Mid domain resembles ligase folds but has substitutions that likely prevent ATP binding, supporting an inactive structural role.

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.

References

Peng, X., Zhang, Y., Sun, L., Peng, R., Zhao, Y., Chen, Y., Lu, X., Yang, H., & Rao, Z. (2023). Structure of the monkeypox virus DNA polymerase holoenzyme. Science, 380(6652), 703–709. https://doi.org/10.1126/science.ade6360