title: DNA3'pp5'G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition.
abstract: DNA3'pp5'G caps synthesized by the 3'-PO4/5'-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3'-OH ends. Aprataxin, an enzyme that repairs A5'pp5'DNA ends formed during abortive ligation by classic 3'-OH/5'-PO4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA3'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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Chauleau
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Agata
authors.initials: A
authors.lastname: Jacewicz
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D029702:Schizosaccharomyces pombe Proteins; D006151:Guanosine; D004247:DNA; D005090:Exodeoxyribonucleases; C569391:Hnt3 protein, S pombe