Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Methods Enzymol. 2024;705:271-309. doi: 10.1016/bs.mie.2024.08.002. Epub 2024 Aug 28.
In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.
在人类中，DNA 聚合酶 δ（pol δ）全酶由 pol δ 和增殖细胞核抗原（PCNA）组成，在滞后链复制过程中进行 DNA 合成，在先导链 DNA 复制的起始以及大多数主要的 DNA 损伤修复途径中也发挥作用。在这些情况下，pol δ 全酶都在引物/模板（P/T）连接处组装，并通过逐步的过程启动 DNA 合成，该过程涉及 PCNA 夹装载器、复制因子 C，并且取决于 DNA 合成途径，还涉及主要的单链 DNA 结合蛋白复合物复制蛋白 A（RPA）。在我们实验室的最近一份报告中，我们设计并利用直接、整体Förster 共振能量转移方法来监测由 RPA 结合的 P/T 连接处 pol δ 全酶组装和 DNA 合成起始的瞬态动力学。在本章中，我们详细介绍了原始方法，并讨论了可以用于监测毫秒（ms）时间尺度上快速动力学反应的适应性。本章中描述的所有方法均使用市售的荧光分光光度计，可轻松适用于替代 DNA 聚合酶和 P/T DNA 底物，并允许整合蛋白质翻译后修饰、辅助因子、DNA 共价修饰、辅助因子、酶等。因此，这些方法广泛可用且广泛适用于表征任何依赖 PCNA 的 DNA 合成途径中 DNA 聚合酶全酶组装和 DNA 合成起始。
