Hou Runhua, Hill Thomas M
Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, 58202-9037
Plasmid. 2002 Jan;47(1):36-50. doi: 10.1006/plas.2001.1553.
In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.
在大肠杆菌染色体中，被Tus-Ter复合物或DNA损伤阻滞的DNA复制叉通过涉及RecA和许多其他重组功能的途径重新启动。为了研究重组在处理被Tus-Ter复合物阻滞的复制叉中的作用，在携带Ter质粒（即含有一个定向阻断DNA复制的Ter位点的质粒）的细胞中评估了与重组相关的基因产物的需求。在所测试的大肠杆菌重组功能中，只有recA缺失会在含有Ter质粒的细胞中产生可观察到的表型，即转化效率低下，并且在表达Tus时维持Ter质粒的能力降低。鉴于目前对复制重新启动的理解，对Ter质粒维持受限的最简单解释是在recA tus(+)背景下重新启动质粒复制的能力降低。然而，我们未能检测到recA功能正常和recA缺陷细胞中Tus导致的复制阻滞效率的差异，这表明无法重新启动被阻滞的复制叉不是生长受限的原因，而是由于RecA提供的额外功能。还研究了对Ter质粒维持受限的其他解释，包括质粒多聚化、质粒重排和拷贝数差异。对Ter质粒维持受限最可能的原因是在相对于外部对照测量拷贝数时，recA细胞中的拷贝数减少。可能是RecA功能的丧失导致在Ter位点被阻滞的复制叉处理不当，从而产生易于降解的底物。
