Chauleau Mathieu, Jacewicz Agata, Shuman Stewart
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
Nucleic Acids Res. 2015 Jul 13;43(12):6075-83. doi: 10.1093/nar/gkv501. Epub 2015 May 24.
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.
由3'-磷酸/5'-羟基连接酶RtcB合成的DNA 3'pp5'G帽对DNA 3'-羟基末端的酶促反应有强烈影响。脱嘌呤嘧啶核酸内切酶是一种修复经典3'-羟基/5'-磷酸连接酶在流产连接过程中形成的A5'pp5'DNA末端的酶，它也是一种DNA 3'脱帽酶，可将DNAppG转化为DNA3'p和GMP。通过利用RtcB利用某些GTP类似物合成DNAppN帽的能力，我们发现脱嘌呤嘧啶核酸内切酶可水解次黄苷和6-O-甲基鸟苷帽，但不擅长去除脱氧鸟苷帽。我们报道了脱嘌呤嘧啶核酸内切酶与GMP复合物的1.5埃晶体结构，该结构表明：（i）GMP与AMP在相同位置以相同的反式核苷构象结合；（ii）脱嘌呤嘧啶核酸内切酶与鸟嘌呤的碱基接触比与腺嘌呤更广泛，通过与鸟嘌呤O6、N1、N2碱基边缘的氢键网络。催化残基His147和His149的丙氨酸突变消除了DNAppG脱帽活性，这表明3'脱鸟苷酰化和5'脱腺苷酰化反应遵循相同的通过共价脱嘌呤嘧啶核酸内切酶-（His147）-NMP中间体的核苷酸转移途径。协调鸟苷核糖羟基的Asp63的丙氨酸突变会损害DNAppG脱帽。
