A novel class of mutations that affect DNA replication in E-coli
A novel class of mutations that affect DNA replication in E-coli
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DOI：
10.1111/j.1365-2958.2007.05651.x
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发表时间：
2007-04-01
影响因子：
3.6
通讯作者：
Wright, Andrew
中科院分区：
文献类型：
--
作者：
Nordman, Jared;Skovgaard, Ole;Wright, Andrew
含有冷敏感性dnaA（cos）等位基因的细胞中DNA复制的过度起始已显示导致广泛的DNA损伤，这可能是由于头对尾复制叉碰撞，其最终导致复制叉崩溃、生长停滞和/或细胞死亡。基于cos突变体的冷敏感表型的抑制因子应该包括影响DNA复制的效率和/或调节的突变的假设，我们对dnaA（cos）突变体菌株进行转座子诱变，并选择可以在30 ℃下形成菌落的突变体衍生物。四个抑制剂的dnaA（cos）介导的冷敏感性进行了鉴定和进一步的特点。基于起点与末端的比率，每个细胞的染色体含量，通过流式细胞术测量，和复制叉抑制剂的敏感性，羟基脲，抑制剂分为两个不同的类别：那些直接抑制过度启动的DNA复制和那些独立的启动。降低DNA聚合酶的chi亚基HolC的细胞水平或ndk（核苷二磷酸激酶）功能丧失的突变属于后一类。我们建议，这些新的抑制突变的功能，通过降低体内复制叉运动的效率，通过减少的情况下，HolC的DNA聚合酶亚基的动态交换，或通过改变DNA复制和脱氧核苷三磷酸合成之间的平衡的情况下，ndk。此外，我们的研究结果表明，过启动和复制叉抑制的敏感性之间的直接相关性，由羟基脲，支持的模型增加头到尾的复制叉碰撞由于过启动。
Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30 degrees C. Four suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those that directly inhibit over-initiation of DNA replication and those that act independently of initiation. Mutations that decrease the cellular level of HolC, the chi subunit of DNA polymerase, or loss of ndk (nucleoside diphosphate kinase) function fall into the latter category. We propose that these novel suppressor mutations function by decreasing the efficiency of replication fork movement in vivo, either by decreasing the dynamic exchange of DNA polymerase subunits in the case of HolC, or by altering the balance between DNA replication and deoxynucleoside triphosphate synthesis in the case of ndk. Additionally, our results indicate a direct correlation between over-initiation and sensitivity to replication fork inhibition by hydroxyurea, supporting a model of increased head-to-tail replication fork collisions due to over-initiation.