A conserved Tyr residue is required for sugar selectivity in a Pol alpha DNA polymerase.
A conserved Tyr residue is required for sugar selectivity in a Pol alpha DNA polymerase.
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Pol α DNA 聚合酶中的糖选择性需要保守的 Tyr 残基。
DOI：
10.1021/bi0202171
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Konigsberg,William
中科院分区：
文献类型：
--
作者：
Yang,Guangwei;Franklin,Matthew;Li,Jing;Lin,T-C;Konigsberg,William
许多DNA聚合酶选择它们的天然底物，脱氧-而不是核糖核苷三磷酸，选择性大于10000倍。检测了亲本酶（pol α DNA聚合酶家族成员RB 69 DNA聚合酶（gp 43）的外切衍生物）棕榈结构域中高度保守残基Tyr 416在帮助聚合酶区分核糖核苷三磷酸、双脱氧核糖核苷三磷酸和脱氧核糖核苷三磷酸中的作用。亲本酶以与dNTPs相对于ddNTPs大致相同的偏好选择dNTPs相对于rNTPs。用亲本酶和两个突变体Y 416 A和Y 416 F进行预稳态动力学分析。Y 416 A突变体掺入核糖核苷酸残基比亲本酶更有效，而Y 416 F突变体比Y 416 A突变体或亲本酶对ddNTP与rNTP利用更宽容。我们还发现，dCDP和rCDP抑制dCTP掺入的Y 416 A突变体，而只有dCDP，而不是rCDP抑制dCTP掺入的亲本酶和Y 416 F突变体。亲本酶以及Y 416 A和Y 416 F突变体都能够将araCTP（1-β-d-阿拉伯呋喃糖基胞嘧啶-5 '-三磷酸）添加到引物中，但相对于dCTP，效率降低。基于我们的动力学结果，在RB 69 gp 43三元复合物的晶体结构的背景下解释，我们认为糖歧视主要是由Tyr 416侧链提供的，其可以在空间上阻断传入rNTP的2 '-OH基团。
Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10 000-fold. The function of a highly conserved residue, Tyr416, in the palm domain of the parental enzyme, an exo-derivative of RB69 DNA polymerase (gp43), a member of the pol α DNA polymerase family, was examined for its role in helping the polymerase discriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. The parental enzyme selected dNTPs vs rNTPs with about the same preference as dNTPs vs ddNTPs. Pre-steady-state kinetic analysis was carried out with the parental enzyme and two mutants, Y416A and Y416F. The Y416A mutant incorporated ribonucleotide residues much more efficiently than the parental enzyme, whereas the Y416F mutant was more permissive toward ddNTP vs rNTP utilization than either the Y416A mutant or the parental enzyme. We also found that both dCDP and rCDP inhibited dCTP incorporation by the Y416A mutant, while only dCDP but not rCDP inhibited dCTP incorporation by the parental enzyme and the Y416F mutant. The parental enzyme and the Y416A and Y416F mutants were all able to add araCTP (1-β-d-arabinofuranosylcytosine-5‘-triphosphate) to a primer but with reduced efficiency relative to dCTP. Based on our kinetic results, interpreted in the context of the crystal structure of the RB69 gp43 ternary complex, we suggest that sugar discrimination is provided mainly by the Tyr416 side chain which can sterically block the 2‘-OH group of an incoming rNTP.