DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.
DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.
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DNA 聚合酶光探针 2-[(4-叠氮苯甲酰基)硫基]-2-脱氧腺苷 5-三磷酸标记大肠杆菌 DNA 聚合酶 I Klenow 片段底物结合位点。
DOI：
10.1021/bi952515m
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Doughty,MB
中科院分区：
文献类型：
--
作者：
Moore2nd,BM;Jalluri,RK;Doughty,MB
核苷酸光探针2-[（4-叠氮苯酰基）硫]-2 ' -脱氧腺苷5 ' -三磷酸(1)被评价为DNA聚合酶I kleow片段的光亲和标记。Photolabel [3H]-1在300 nm处对Klenow片段进行光解共价标记，在5.7 μM处以约1:1的摩尔比达到饱和，EC50（最大光掺入50%时的有效浓度）约为0.74 μM。饱和浓度的聚（dA）·(T)10保护了Klenow片段免受[3H]-1的光结合，而近似等于其自由酶形式的TTP浓度将[3H]-1光结合的剂量-响应曲线移动了2倍，表明TTP和1之间存在竞争关系。此外，[3H]-1在Klenow片段中的光结合是绝对需要镁的，在没有镁的情况下，在10 μM的浓度下没有观察到明显的光结合。这些结果表明，正如设计的那样，photoprobe1与dNTP和Klenow片段上的部分模板引物结合位点结合。对Klenow片段进行光亲和标记，得到一个放射性标记的色氨酸片段，该片段通过HPLC分离得到；序列分析鉴定出asp732 - ile733 - his734 - arg735片段中的asp732为共价修饰位点。分子模型和互补核磁共振分析表明，C3 ' -exo和C2 ' -exo-C3 ' -endo对称扭曲呋喃糖环折叠物具有高反碱构象和+sc C-5扭角。利用asp732作为光标记上叠氮化物α-氮锚点的对接研究表明，dNTP结合位点位于DNA结合间隙的边缘，与外切酶位点相对，模板结合位点包括Klenow片段手指基序中的螺旋O。
The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2‘-deoxyadenosine 5‘-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 μM and with an EC50(the effective concentration at 50% maximum photoincorporation) of about 0.74 μM. Saturating concentrations of poly(dA)·(T)10protect the Klenow fragment from [3H]-1photoincorporation, and TTP at a concentration approximately equal to itsKDfor the free enzyme form shifts the dose−response curve for photoincorporation of [3H]-1into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and1. Additionally, the photoincorporation of [3H]-1into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of1up to 10 μM in the absence of magnesium. These results demonstrate that, as designed, photoprobe1binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by1yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732in the peptide fragment Asp732-Ile733-His734-Arg735as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of1indicated preferred C3‘-exo and C2‘-exo-C3‘-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732as an anchor point for the azide α-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.