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通过单分子研究解析细菌DNA复制中的DnaB解旋酶动力学。

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies.

作者信息

Spinks Richard R, Spenkelink Lisanne M, Stratmann Sarah A, Xu Zhi-Qiang, Stamford N Patrick J, Brown Susan E, Dixon Nicholas E, Jergic Slobodan, van Oijen Antoine M

机构信息

Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales 2522, Australia.

Illawarra Health & Medical Research Institute, Wollongong, New South Wales 2522, Australia.

出版信息

Nucleic Acids Res. 2021 Jul 9;49(12):6804-6816. doi: 10.1093/nar/gkab493.

Abstract

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.

摘要

在大肠杆菌中,DnaB解旋酶构成了DNA复制复合体组装的基础。DnaB在复制叉处的稳定性对于成功启动和进行复制可能很重要。单分子实验通过表明复制机器的组件会与环境中的游离分子交换,显著改变了高度稳定的复制机器的经典模型。然而,由于技术限制,目前缺乏在复制背景下对DnaB稳定性的准确评估。我们利用体外荧光单分子成像技术,观察加载在叉状DNA模板上的DnaB。这些解旋酶在复制叉处高度稳定,其观察到的驻留时间约为30分钟。添加其余的复制因子会导致单个DnaB解旋酶整合到活性复制体中,成为其一部分。与其他复制体组件的动态行为不同,DnaB在整个复制过程中都维持在复制体内。有趣的是,我们观察到额外的解旋酶与复制叉存在短暂相互作用。这种相互作用依赖于钳位加载复合体的τ亚基。总的来说,我们的单分子观察结果巩固了DnaB解旋酶作为复制体稳定锚定物的作用,但也揭示了其进行动态相互作用的能力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3963/8266626/1b947f127415/gkab493gra1.jpg

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