DNA3'pp5'G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition.
Bottom Line: We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge.Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3' de-guanylylation and 5' de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)-NMP intermediate.Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.
Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.Show MeSH
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Mentions: In brief, the aprataxin-DNA interface in the present structure spans 8-nt (Figure 7). The duplex end is bookmarked by Phe34, which makes a π-stack on the guanine nucleobase of the 5′ G:C base pair. Phe65 packs again the penultimate ribose of the substrate strand. All other contacts are with the phosphate backbone. Lys67 and His165 coordinate the terminal phosphodiester of the substrate strand and Lys161 coordinates the second phosphodiester. Distal atomic contacts to the template strand are via the Zn-finger domain. Arg209 makes a bidentate interaction with the sixth phosphodiester. The seventh phosphodiester is engaged by the Phe211 and Thr212 main-chain amides. The eight phosphodiester receives a hydrogen bond from by the Thr212 side chain (Figure 7).
Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.