Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Biochemistry. 2010 May 4;49(17):3545-54. doi: 10.1021/bi100042b.
This study examines the role of the p12 subunit in the function of the human DNA polymerase delta (Pol delta) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the four-subunit complex, the three-subunit complex lacking p12, and site-directed mutants of each lacking proofreading exonuclease activity. Results show that p12 modulates the rate and fidelity of DNA synthesis by Pol delta. All four complexes synthesize DNA in a rapid burst phase and a slower, more linear phase. In the presence of p12, the burst rates of DNA synthesis are approximately 5 times faster, while the affinity of the enzyme for its DNA and dNTP substrates appears unchanged. The p12 subunit alters Pol delta fidelity by modulating the proofreading 3' to 5' exonuclease activity. In the absence of p12, Pol delta is more likely to proofread DNA synthesis because it cleaves single-stranded DNA twice as fast and transfers mismatched DNA from the polymerase to the exonuclease sites 9 times faster. Pol delta also extends mismatched primers 3 times more slowly in the absence of p12. Taken together, the changes that p12 exerts on Pol delta are ones that can modulate its fidelity of DNA synthesis. The loss of p12, which occurs in cells upon exposure to DNA-damaging agents, converts Pol delta to a form that has an increased capacity for proofreading.
本研究通过比较四聚体复合物、缺乏 p12 的三聚体复合物以及每个缺乏校对外切酶活性的定点突变体催化的 DNA 合成和降解动力学，研究了 p12 亚基在人类 DNA 聚合酶 δ（Pol δ）全酶功能中的作用。结果表明，p12 通过 Pol δ 调节 DNA 合成的速率和保真度。所有四个复合物均在快速爆发阶段和较慢的、更线性的阶段合成 DNA。在 p12 存在的情况下，DNA 合成的爆发速率大约快 5 倍，而酶对其 DNA 和 dNTP 底物的亲和力似乎没有变化。p12 亚基通过调节校对 3' 到 5' 外切酶活性来改变 Pol δ 的保真度。在缺乏 p12 的情况下，Pol δ 更有可能校对 DNA 合成，因为它切割单链 DNA 的速度快两倍，将错配的 DNA 从聚合酶转移到外切酶位点的速度快 9 倍。在缺乏 p12 的情况下，Pol δ 也会将错配引物延伸得慢三倍。总之，p12 对 Pol δ 施加的变化可以调节其 DNA 合成的保真度。p12 的缺失（在细胞暴露于 DNA 损伤剂时发生）将 Pol δ 转化为一种具有增强校对能力的形式。
