Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Institut National de la Recherche Agronomique, Génétique Microbienne, 78350 Jouy-en-Josas, France.
Open Biol. 2017 Sep;7(9). doi: 10.1098/rsob.170146.
During replication two replicative polymerases function at the replisome to collectively carry out genome replication. In a reconstituted replication assay, PolC is the main polymerase while the lagging strand DnaE polymerase briefly extends RNA primers synthesized by the primase DnaG prior to handing-off DNA synthesis to PolC. Here, we show that (i) the polymerase activity of DnaE is essential for both the initiation and elongation stages of DNA replication, (ii) its error rate varies inversely with PolC concentration, and (iii) its misincorporations are corrected by the mismatch repair system post-replication. We also found that the error rates in cells encoding mutator forms of both PolC and DnaE are significantly higher (up to 15-fold) than in PolC mutants. , we showed that (i) the polymerase activity of DnaE is considerably stimulated by DnaN, SSB and PolC, (ii) its error-prone activity is strongly inhibited by DnaN, and (iii) its errors are proofread by the 3' > 5' exonuclease activity of PolC in a stable template-DnaE-PolC complex. Collectively our data show that protein-protein interactions within the replisome modulate the activity and fidelity of DnaE, and confirm the prominent role of DnaE during replication.
在复制过程中，两个复制聚合酶在复制体上协同作用，共同进行基因组复制。在重组复制实验中，PolC 是主要的聚合酶，而滞后链 DnaE 聚合酶在将 RNA 引物的 DNA 合成移交给 PolC 之前，短暂地延伸由 DnaG 引发酶合成的 RNA 引物。在这里，我们表明：（i）DnaE 的聚合酶活性对 DNA 复制的起始和延伸阶段都是必不可少的；（ii）其错误率与 PolC 浓度成反比；（iii）其错误掺入物在复制后由错配修复系统进行校正。我们还发现，编码 PolC 和 DnaE 突变体的细胞中的错误率（高达 15 倍）明显高于 PolC 突变体。此外，我们表明：（i）DnaN、SSB 和 PolC 极大地刺激了 DnaE 的聚合酶活性；（ii）DnaN 强烈抑制其易错活性；（iii）其错误由 PolC 的 3' > 5' 外切核酸酶活性在稳定的模板-DnaE-PolC 复合物中进行校对。总之，我们的数据表明，复制体中的蛋白-蛋白相互作用调节了 DnaE 的活性和保真度，并证实了 DnaE 在复制过程中的突出作用。
