A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present.大肠杆菌（Escherichia coli）的翻译起始因子2（translation initiation factor 2, IF2）在体外参与噬菌体Mu（bacteriophage Mu）转座过程时，展现出一项此前未被报道的意外功能：可介导重组反应向复制过程的过渡，进而通过复制重启蛋白（replication restart proteins）启动复制。该功能提示IF2可能参与细胞内的重启机制，并调控基因组完整性（genome integrity）的维持。为探究IF2对重启机制的潜在影响，我们通过甲基磺酸甲酯（methyl methanesulfonate, MMS）与紫外线（UV）诱导DNA损伤，表征了IF2对细胞恢复能力的影响。阻断全长IF2-1表达，或抑制截短型IF2-2、IF2-3同工型（isoforms）表达的突变株，对DNA损伤后的细胞生长或恢复的影响存在显著差异，且分别作用于不同的重启机制。仅表达IF2-2/3的缺失突变株（del1）在DNA损伤剂MMS处理下，表现出严重的生长敏感性。该突变株在移除MMS后，其DNA损伤修复与复制重启能力与野生型菌株无明显差异，但在MMS持续存在时无法维持细胞生长；不过，通过破坏编码复制叉停滞时诱导的细胞分裂抑制剂的sulA基因，可部分恢复该突变株在MMS中的生长能力。此外，del1对MMS的敏感表型与解旋酶缺陷型重启蛋白突变株priA300一致。上位性分析（epistasis analysis）显示，del1与priA300联合突变后，并未对细胞经MMS与UV处理后的恢复能力产生额外影响；但仅允许IF2-1表达的del2/3突变株，与priA300联合时会协同增强UV敏感性。本研究结果表明，全长IF2具备与截短型同工体不同的功能，可影响依赖PriA解旋酶的重启功能的参与或活性，从而使细胞在DNA损伤剂存在时仍能维持正常生长。