title: How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.
abstract: A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.
authors.affiliation: Institute of Biological Chemistry, and ‡Genomics Research Center, Academia Sinica , 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan.
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Wu
authors.forename: Mei-I
authors.initials: MI
authors.lastname: Su
authors.forename: Jian-Li
authors.initials: JL
authors.lastname: Wu
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: Liang-Hin
authors.initials: LH
authors.lastname: Lim
authors.forename: Chun-Wei Eric
authors.initials: CW
authors.lastname: Wang
authors.forename: Frank H T
authors.initials: FH
authors.lastname: Nelissen
authors.forename: Ming-Chuan Chad
authors.initials: MC
authors.lastname: Chen
authors.forename: Jurgen F
authors.initials: JF
authors.lastname: Doreleijers
authors.forename: Sybren S
authors.initials: SS
authors.lastname: Wijmenga
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D000357:African Swine Fever / Q000821:virology; D000358:African Swine Fever Virus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013552:Swine / Q000821:virology
chemical_list: D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; C109534:DNA polymerase X; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase