Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication.
Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication.
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DOI：
10.1016/bs.mie.2022.03.011
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发表时间：
2022
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基因组复制是由一个多蛋白质复合体（称为复制体）中酶的高度调节活性完成的。两种主要的酶，DNA聚合酶和解旋酶，催化亲本DNA双链体的前导链上的连续DNA合成，而滞后链不连续地合成。解旋酶和DNA聚合酶本身对于解旋/复制双链DNA来说是催化效率低且弱的马达。然而，当解旋酶和DNA聚合酶在功能上和物理上偶联时，它们催化快速和高度进行性的前导链DNA合成。DNA聚合酶具有3 '-5'核酸外切酶活性，其去除错误掺入新生DNA中的核苷酸。DNA合成动力学、持续合成能力和准确性受复制体内解旋酶、DNA聚合酶和核酸外切酶活性的相互作用控制。本章介绍了定量的生物化学和生物物理学方法来研究DNA复制过程中这三个关键活动的耦合。该方法包括实时定量的动力学的DNA解旋合成的耦合解旋酶-DNA聚合酶复合物，2-氨基嘌呤荧光为基础的测定映射的精确位置的解旋酶和DNA聚合酶相对于复制叉交界处，和放射性测定，以研究耦合的DNA聚合酶，核酸外切酶，解旋酶的活动过程中的前导链DNA合成。本文以噬菌体T7复制蛋白为例介绍了这些方法，但经过适当修改后也可应用于其他系统。
Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA. However, when a helicase and DNA polymerase are functionally and physically coupled, they catalyze fast and highly processive leading strand DNA synthesis. DNA polymerase has a 3’-5’ exonuclease activity, which removes nucleotides misincorporated in the nascent DNA. DNA synthesis kinetics, processivity, and accuracy are governed by the interplay of the helicase, DNA polymerase, and exonuclease activities within the replisome. This chapter describes quantitative biochemical and biophysical methods to study the coupling of these three critical activities during DNA replication. The methods include real-time quantitation of kinetics of DNA unwinding-synthesis by a coupled helicase-DNA polymerase complex, a 2-aminopurine fluorescence-based assay to map the precise positions of helicase and DNA polymerase with respect to the replication fork junction, and a radiometric assay to study the coupling of DNA polymerase, exonuclease, and helicase activities during processive leading strand DNA synthesis. These methods are presented here with bacteriophage T7 replication proteins as an example but can be applied to other systems with appropriate modifications.