Catalano C E, Allen D J, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1990 Apr 17;29(15):3612-21. doi: 10.1021/bi00467a004.
The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.
叠氮脱氧核糖核酸双链体的合成及其用于光标记DNA聚合酶的方法先前已有描述（吉布森和本科维奇，1987年）。我们现在展示利用这种叠氮脱氧核糖核酸光探针作为大肠杆菌DNA聚合酶I（克列诺片段）的底物以及对该蛋白质进行光亲和标记的详细实验。叠氮脱氧核糖核酸双链体是该酶聚合酶和3'→5'核酸外切酶活性的有效底物。然而，含叠氮碱基的水解降解显著受损。基于这些双链体对该酶进行光标记的能力，我们已确定蛋白质与双链DNA的五到七个碱基之间存在接触。在镁存在的情况下，将叠氮脱氧核糖核酸与克列诺片段一起温育会导致原位形成一个模板引物，其中含叠氮碱基结合在该酶的聚合酶催化位点。对该复合物进行光解，随后进行蛋白酶消化并分离DNA标记的肽段，从而鉴定出一个被光反应性DNA底物修饰的单一残基。我们确定酪氨酸766是被修饰的氨基酸，从而在蛋白质中定位聚合反应的催化位点。已制备了一种甘露糖基标记的DNA双链体作为蛋白质结构的荧光探针。这已被用于确定与克列诺片段结合时引物末端的位置。当双链体的引物链中含有五个未配对碱基时，引物末端主要位于该酶的核酸外切酶催化位点。通过该酶的核酸外切酶活性去除错配碱基会产生一种二元复合物，此时引物末端主要结合在聚合酶活性位点。所呈现的数据表明，该酶核酸外切酶活性中的限速步骤是引物末端从聚合酶催化位点向核酸外切酶催化位点的易位。
