Flåtten Ingvild, Helgesen Emily, Pedersen Ida Benedikte, Waldminghaus Torsten, Rothe Christiane, Taipale Riikka, Johnsen Line, Skarstad Kirsten
Department of Molecular Cell Biology and Department of Microbiology, Oslo University Hospital, Oslo, Norway.
Philipps-Universität Marburg, LOEWE Center for Synthetic Microbiology-SYNMIKRO, Chromosome Biology Group, Marburg, Germany.
J Bacteriol. 2017 Nov 14;199(24). doi: 10.1128/JB.00412-17. Print 2017 Dec 15.
The mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation The gene for the clamp loader subunits τ and γ, , but not the mutant , was found to suppress (Ts) when overexpressed. Purified mutant protein was found to be functional , and few phenotypes were found apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at never reach the terminus in mutant cells. The SOS response was found to be induced, and a combination of the mutation with and mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of and , respectively. The results indicate that the mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the -encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. The clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.
该突变是在对温度敏感的拓扑异构酶IV突变的多拷贝抑制子进行筛选时发现的。发现钳位装载亚基τ和γ的基因（而非突变体）在过表达时可抑制温度敏感（Ts）。纯化的突变蛋白具有功能，除了在丰富培养基中DNA分配存在问题外，未发现其他表型。我们在此表明，在突变细胞中，大量在起始的复制叉从未到达终点。发现SOS反应被诱导，并且该突变与其他突变的组合导致活力降低。分别使和失活后，突变细胞表现出广泛的染色体片段化和降解。结果表明，突变细胞存在断裂的复制叉，这些复制叉需要通过同源重组进行修复。我们认为，钳位装载器的编码τ和γ亚基或钳位装载器复合物本身在无需广泛同源重组的情况下重新启动停滞的复制叉中发挥作用。钳位装载器复合物在协调复制叉处复制体的活性以及为滞后链合成装载β钳位方面发挥作用。复制叉经常遇到障碍，如模板损伤、二级结构和紧密结合的蛋白质复合物，这会导致复制叉停滞。一些复制叉重新启动的途径已得到表征，但对于其中涉及的因素和机制仍有很多未知之处。我们在这项工作中对钳位装载器突变体进行了表征。我们发现，突变的钳位装载器无法以适当方式应对复制叉遇到的天然障碍，并提出钳位装载器在重新启动停滞的复制叉中发挥作用。
