García Patty B, Robledo Nicole L, Islas Angel L
Department of Biology, Santa Clara University, Santa Clara, California 95053-0268, USA.
Biochemistry. 2004 Dec 28;43(51):16515-24. doi: 10.1021/bi0491853.
DNA polymerases use an uninterrupted template strand to direct synthesis of DNA. However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction. Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis. The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases. To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I. Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension. We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis. Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides. For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other. These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase. Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.
DNA聚合酶利用一条不间断的模板链来指导DNA的合成。然而，一些DNA聚合酶可以通过结合并使两个不连续的模板并列，从而跨越这两个模板合成DNA，导致在连接点处的合成。具有3'突出端的引物/模板双链体是特别有效的底物，这表明DNA聚合酶将突出端用作模板联会的微同源区域。这些突出端的形成可能是DNA聚合酶进行非模板指导的核苷酸添加的结果。为了研究模板转换的相对程度和机制，我们研究了大肠杆菌DNA聚合酶I的3'-5'核酸外切酶缺陷型大片段进行模板转换和非模板指导的核苷酸添加的体外酶动力学。将非模板指导的核苷酸添加和模板转换与标准引物延伸进行了比较。我们发现，非模板指导的核苷酸添加和模板转换显示出相似的速率，并且比正常的模板指导的DNA合成慢约100倍。此外，非模板指导的核苷酸添加在向DNA末端添加dAMP方面比其他三种核苷酸表现出10倍的偏好。对于模板转换，动力学分析表明，两种模板底物作为一个随机双反应物系统，一种底物对另一种底物的底物结合具有混合型抑制作用。这些数据首次确定了两个不连续的DNA底物与单个DNA聚合酶的结合动力学。我们的结果表明，尽管这些活性相对较弱，但非模板指导的核苷酸添加和模板转换使DNA聚合酶能够以易出错的方式克服模板链中的断裂。
