O'Neal Luke G, Drucker Madeline N, Lai Ngoc Khanh, Clemente Ashley F, Campbell Alyssa P, Way Lindsey E, Hong Sinwoo, Holmes Emily E, Rancic Sarah J, Sawyer Nicholas, Wang Xindan, Thrall Elizabeth S
Department of Chemistry and Biochemistry, Fordham University, Bronx, NY 10458.
Department of Biology, Indiana University, Bloomington, IN 47405.
bioRxiv. 2025 Mar 11:2025.03.10.642433. doi: 10.1101/2025.03.10.642433.
Ring-shaped sliding clamp proteins are essential components of the replication machinery, the replisome, across all domains of life. In bacteria, DNA polymerases bind the sliding clamp, DnaN, through conserved short peptide sequences called clamp-binding motifs. Clamp binding increases the processivity and rate of DNA synthesis and is generally required for polymerase activity. The current understanding of clamp-polymerase interactions was elucidated in the model bacterium , which has a single replicative polymerase, Pol III. However, many bacteria have two essential replicative polymerases, such as PolC and DnaE in . PolC performs the bulk of DNA synthesis whereas the error-prone DnaE only synthesizes short stretches of DNA on the lagging strand. How the clamp interacts with the two polymerases and coordinates their activity is unknown. We investigated this question by combining in vivo single-molecule fluorescence microscopy with biochemical and microbiological assays. We found that PolC-DnaN binding is essential for replication, although weakening the interaction is tolerated with only minimal effects. In contrast, the DnaE-DnaN interaction is dispensable for replication. Altering the clamp-binding strength of DnaE produces only subtle effects on DnaE cellular localization and dynamics, but it has a substantial impact on mutagenesis. Our results support a model in which DnaE acts distributively during replication but can be stabilized on the DNA template by clamp binding. This study provides new insights into the coordination of multiple replicative polymerases in bacteria and the role of the clamp in polymerase processivity, fidelity, and exchange.
环形滑动夹蛋白是所有生命域中复制机器（即复制体）的重要组成部分。在细菌中，DNA聚合酶通过称为夹结合基序的保守短肽序列与滑动夹DnaN结合。夹结合增加了DNA合成的持续合成能力和速率，并且通常是聚合酶活性所必需的。目前对夹-聚合酶相互作用的理解是在模式细菌中阐明的，该细菌具有单一的复制聚合酶Pol III。然而，许多细菌有两种必需的复制聚合酶，如[具体细菌名称]中的PolC和DnaE。PolC负责大部分的DNA合成，而易出错的DnaE仅在滞后链上合成短片段的DNA。夹如何与这两种聚合酶相互作用并协调它们的活性尚不清楚。我们通过将体内单分子荧光显微镜与生化和微生物学分析相结合来研究这个问题。我们发现PolC-DnaN结合对于复制至关重要，尽管减弱这种相互作用仅产生最小影响时仍可被耐受。相比之下，DnaE-DnaN相互作用对于复制是可有可无的。改变DnaE的夹结合强度仅对DnaE的细胞定位和动态产生细微影响，但对诱变有重大影响。我们的结果支持一个模型，即DnaE在复制过程中以分布式方式起作用，但可以通过夹结合在DNA模板上稳定下来。这项研究为细菌中多种复制聚合酶的协调以及夹在聚合酶持续合成能力、保真度和交换中的作用提供了新的见解。
