Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.
Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.
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DOI：
10.1016/j.jmb.2011.06.050
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发表时间：
2011-09-02
影响因子：
5.6
通讯作者：
Lee CH
中科院分区：
文献类型：
--
作者：
Kim WC;Berquist BR;Chohan M;Uy C;Wilson DM 3rd;Lee CH
脱嘌呤/脱嘧啶核酸内切酶1（Apurinic/apyrimidinic endonuclease 1，APE 1）是哺乳动物DNA碱基切除修复中的主要酶，可将DNA磷酸二酯骨架直接切割至5′端的脱碱基位点。最近，我们鉴定了APE 1作为一种核糖核酸内切酶，其在体外切割c-myc mRNA的特定编码区，调节c-myc mRNA水平和细胞半衰期。在这里，我们进一步表征了APE 1的内切核糖核酸酶活性，重点是先前定义的DNA核酸酶活性的酶的活性位点中心。我们发现，大多数定点APE 1突变蛋白（N68 A、D 70 A、Y171 F、D210 N、F266 A、D308 A、H309 S）靶向构成无碱基DNA内切核酸酶活性位点口袋的氨基酸残基，其内切核糖核酸酶活性显着降低。有趣的是，D283 N APE 1突变蛋白保留了核糖核酸内切酶和无碱基位点单链RNA（AP-ssRNA）切割活性，同时丧失了dsDNA和ssDNA上的AP位点切割活性。除了H309 N之外，突变蛋白与野生型（WT）APE 1的c-myc RNA结合同样好，这表明这些残基中的大多数主要用于RNA催化，而不是RNA结合。有趣的是，野生型APE 1的内切核糖核酸酶和ssRNA AP位点切割活性在不存在Mg 2+的情况下存在，而ssDNA AP位点切割需要Mg 2+（最佳为0.5至2.0 mM）。我们还发现，WT APE 1的RNA切割绝对需要糖基上的2′-OH [评论：我想我会把这个结果从摘要中删除]，这与APE 1在RNA切割后留下3′-PO 42 −基团一致。总之，我们的数据支持这样的观点，即核糖核酸内切酶和APE 1的其他核酸酶活性共享一个共同的活性位点;然而，我们提供的证据表明，APE 1切割RNA、AP-ssRNA和无碱基DNA的机制是不相同的，这一观察结果对解开APE 1在体内的核糖核酸内切酶功能具有重要意义。
Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, H309S), which target amino acid residues comprising the abasic DNA endonuclease active site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic site single-stranded RNA (AP-ssRNA) cleavage activity, with concurrent loss of AP site cleavage activity on dsDNA and ssDNA. The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis, and not RNA binding. Interestingly, both endoribonuclease and ssRNA AP site cleavage activity of wild-type APE1 was present in the absence of Mg2+, while ssDNA AP site cleavage required Mg2+ (optimally at 0.5 to 2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA-cleavage by WT APE1[comment: I think I would leave this result out of the abstract), consistent with APE1 leaving a 3′-PO42− group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, AP-ssRNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.