letters 942 nature structural biology • volume 8 number 11 • november 2001 Solution structure of a viral DNA polymerase X and evidence for a mutagenic function Alexander K. Showalter 1 , In-Ja L. Byeon 2 , Mei-I Su 1 and Ming-Daw Tsai 1–4 1 Department of Chemistry, 2 The Campus Chemical Instrument Center, 3 Department of Biochemistry and 4 The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA. The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide poly- merases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is impor- tant for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may con- tribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator. A number of DNA polymerases have recently been described that copy template DNA with very low fidelity. The biological relevance of this error-prone activity, however, remains uncer- tain. Among the more extreme examples of an error-prone poly- merase is African swine fever virus DNA polymerase X (ASFV Pol X). Pol X is the only polymerase that catalyzes nucleotide mispair formation with comparable efficiency to that with which it catalyzes formation of all four Watson-Crick base pairs 1 . It is also the only known extreme low fidelity polymerase encoded by a virus. On the basis of such properties, Pol X has been postulat- ed to function as a strategic DNA mutase, promoting viral hypervariability via low fidelity synthesis 1 . Mammalian DNA polymerase β (Pol β), the known protein with the highest sequence homology to Pol X, functions in base excision repair (BER) 2 . The last step of BER is sealing of the newly synthesized, repaired strand into the genome by a DNA ligase. Based on its sequence homology to Pol β 3 and an apparent gap-filling functionality 1,4 , Pol X is believed to participate in a viral BER pathway. This, in addition to the observations outlined above, led to the proposal that viral BER is mutagenic, driven by the error-prone activity of Pol X 1 . Here we report the structure of Pol X. In addition, a unique tolerance of the bulky G-G mismatch by ASFV DNA ligase is demonstrated, consistent with the hypothesis of a mutagenic viral BER pathway. Structure of Pol X The structure of Pol X was determined by heteronuclear NMR methods by assigning >97% backbone and >85% side chain res- onances, and by simulated-annealing calculations using a total of 2,597 NMR-derived restraints. The structure is well defined Fig. 1 NMR solution structure of Pol X and structural comparison with Pol β. a, Backbone traces of the ensemble of 20 simulated annealing (SA) structures of Pol X. b, Ribbon diagram of Pol X with secondary structural elements. The minimized mean structure is used. The side chains of the catalytic Asp triad are shown in purple, and the disulfide bond is in dark blue. c, Ribbon diagram of Pol β with secondary structural elements and with the 8 kDa domain and thumb subdomain omitted (adapted from PDB code 1BPB). The side chains of the catalytic Asp triad are shown in purple. d, Structure-based sequence alignment of Pol X with the palm and fingers subdomains of Pol β. Secondary structural elements as defined by the NMR structure of Pol X are shown above the alignment, and those by the crystal structure of Pol β (PDB code 1BPB) are shown below. Identical residues are labeled in yellow, and the catalytic Asp tri- ads are labeled in purple. In all structural figures, the letters ‘F’, ‘P’ and ‘T’ refer to the fingers, palm and thumb subdomain, respectively. a b c d © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com