Nanfara Michael T, Babu Vignesh M P, Ghazy Mohamed A, Sutton Mark D
Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, 14214, United States of America.
Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt.
PLoS One. 2016 Sep 29;11(9):e0163643. doi: 10.1371/journal.pone.0163643. eCollection 2016.
The E. coli dnaN-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.
大肠杆菌中由dnaN编码的β滑动夹蛋白在调控5种细菌DNA聚合酶、错配修复相关蛋白以及其他几种参与DNA复制的蛋白对DNA的作用方面发挥着关键作用。体外实验结果表明，β夹加载到DNA上既依赖于DnaX夹加载复合物，也依赖于几种离散的滑动夹与DNA的相互作用。然而，这些DNA相互作用对大肠杆菌生存能力的重要性，以及β夹支持其众多伙伴蛋白作用的能力，尚未得到研究。为了确定β夹与DNA的相互作用对大肠杆菌应对不同类型DNA损伤能力的贡献，我们使用丙氨酸扫描对22个单独的残基进行突变，这些残基位于已知或靠近已知与DNA模板接触的3个不同的β夹表面，包括P20 - L27残基（此处称为环I）、H148 - Y154残基（环II）以及β夹中心孔周围的7个不同残基，DNA模板从中穿过。这22个dnaN突变体中有20个支持细菌生长。虽然这20个突变体中没有一个对过氧化氢或紫外线敏感，但有12个对萘啶酸敏感，5个对甲基磺酸甲酯敏感，8个在DNA损伤诱导的诱变频率上有适度改变，2个可能在支持hda功能方面受损。综上所述，这些结果表明，离散的β夹与DNA相互作用区域有助于大肠杆菌耐受特定类型的DNA损伤。
