Lam Wai-Chung, Thompson Elizabeth H Z, Potapova Olga, Sun Xiaojun Chen, Joyce Catherine M, Millar David P
Department of Molecular Biology, MB-19, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2002 Mar 26;41(12):3943-51. doi: 10.1021/bi0120603.
The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting.
运用生化和光谱技术相结合的方法，对DNA聚合酶I的Klenow片段的3'-5'核酸外切酶活性机制进行了研究。通过定点诱变，对与可切割磷酸二酯键5'侧的DNA底物相互作用的侧链进行丙氨酸取代。为了探究所选侧链在核酸外切酶反应中的作用，测定了每种突变蛋白对单链和双链DNA底物进行3'-5'核酸外切酶切割的动力学参数。结果表明，与倒数第二个核苷酸相互作用的侧链（Q419、N420和Y423）对于将DNA底物锚定在活性位点或确保可切割磷酸的正确几何形状很重要。相比之下，与DNA末端第三个核苷酸相互作用的侧链（K422和R455）不直接参与单链DNA的核酸外切酶切割。Q419、Y423和R455的丙氨酸取代对单链和双链DNA的切割有明显不同的影响，在双链底物的情况下导致活性损失更大。用丹磺酰标记的引物/模板进行的时间分辨荧光各向异性衰减测量表明，Q419A、Y423A和R455A突变破坏了Klenow片段使双链DNA解链并在核酸外切酶位点结合磨损末端的能力。相比之下，N420A突变稳定了双链末端与核酸外切酶位点的结合，这表明N420侧链通过在结合的DNA底物中引入张力来促进3'-5'核酸外切酶反应。总之，这些结果表明，与末端第二个或第三个核苷酸相互作用的蛋白质侧链可以通过将底物锚定在活性位点或确保可切割磷酸的正确几何形状参与核酸外切酶反应的化学步骤，并且通过促进双链解链参与双链DNA水解的化学前步骤。
