DNA3′pp5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

• Published in: Nucleic acids research 2015-07, Vol.43 (12), p.6075-6083
• Main Authors: Chauleau, Mathieu, Jacewicz, Agata, Shuman, Stewart
• Format: Article
• Language: English
• Subjects: Nucleic Acid Enzymes
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv501

DNA 3′ pp 5′ G caps synthesized by the 3′-PO 4 /5′-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3′-OH ends. Aprataxin, an enzyme that repairs A 5′ pp 5′ DNA ends formed during abortive ligation by classic 3′-OH/5′-PO 4 ligases, is also a DNA 3′ de-capping enzyme, converting DNAppG to DNA 3′ p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. 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.