The mutagenic footprint of low-fidelity Pol I ColE1 plasmid replication in E. coli reveals an extensive interplay between Pol I and Pol III.
The mutagenic footprint of low-fidelity Pol I ColE1 plasmid replication in E. coli reveals an extensive interplay between Pol I and Pol III.
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DOI：
10.1007/s00294-013-0415-9
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发表时间：
2014-08
期刊：
影响因子：
2.5
通讯作者：
Camps, Manel
中科院分区：
文献类型：
--
作者：
Troll, Christopher;Yoder, Jordan;Alexander, David;Hernandez, Jaime;Loh, Yueling;Camps, Manel
ColE1质粒的复制是单向的，需要两种DNA聚合酶：DNA聚合酶I （Pol I）和DNA聚合酶III （Pol III）。Pol I通过延伸RNA引物启动前导链合成，允许Pol III全酶组装并完成两条链的复制。本研究的目的是研究ColE1质粒复制过程中Pol I和Pol III之间的相互作用，以期对Pol I在体内的功能有新的认识。我们的方法包括在ColE1质粒复制过程中使用Pol I的低保真突变体（LF-Pol I）产生的突变作为Pol I复制的足迹。这种方法允许在质粒上以高分辨率绘制Pol I复制区域。此外，我们能够估计整个质粒中Pol I突变的链结性，从而使我们能够估计LF-Pol I在体内的谱。我们的研究产生了以下三个机制见解：1)我们确定了聚合酶开关可能位于复制起始下游约200 bp的位置；2)我们发现证据表明Pol I可以复制两条链，这支持了先前关于Pol I和Pol III之间功能冗余的研究。3)我们发现证据指向Pol I在延迟链复制终止过程中的特定作用。此外，我们还说明了我们的链特异性足迹方法如何用于剖析体内调节Pol I保真度的因素。
ColE1 plasmid replication is unidirectional and requires two DNA polymerases: DNA polymerase I (Pol I) and DNA polymerase III (Pol III). Pol I initiates leading-strand synthesis by extending an RNA primer, allowing the Pol III holoenzyme to assemble and to finish replication of both strands. The goal of the present work is to study the interplay between Pol I and Pol III during ColE1 plasmid replication, in order to gain new insights into Pol I function in vivo. Our approach consists of using mutations generated by a low fidelity mutant of Pol I (LF-Pol I) during replication of a ColE1 plasmid as a footprint for Pol I replication. This approach allowed mapping areas of Pol I replication on the plasmid with high resolution. In addition, we were able to approximate the strandedness of Pol I mutations throughout the plasmid, allowing us to estimate the spectrum of the LF-Pol I in vivo. Our study produced the following three mechanistic insights: 1) we identified the likely location of the polymerase switch at ~200 bp downstream of replication initiation; 2) we found evidence suggesting that Pol I can replicate both strands, supporting earlier studies indicating a functional redundancy between Pol I and Pol III 3) we found evidence pointing to a specific role of Pol I during termination of lagging-strand replication. In addition, we illustrate how our strand-specific footprinting approach can be used to dissect factors modulating Pol I fidelity in vivo.
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DOI：
10.1016/j.bbapap.2009.06.010
发表时间：
2010-05
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Sutton MD
通讯作者：
Sutton MD
影响因子：
3.7
作者：
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通讯作者：
Webb JS
DOI：
10.1073/pnas.1210309109
发表时间：
2012-10-09
影响因子：
11.1
作者：
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通讯作者：
Foster, Patricia L.
影响因子：
3.2
作者：
MAKI, H;BRYAN, SK;MOSES, RE
通讯作者：
MOSES, RE
影响因子：
14.9
作者：
Allen JM;Simcha DM;Ericson NG;Alexander DL;Marquette JT;Van Biber BP;Troll CJ;Karchin R;Bielas JH;Loeb LA;Camps M
通讯作者：
Camps M