Ganai Rais A, Bylund Göran O, Johansson Erik
Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden.
Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden
Nucleic Acids Res. 2015 Jan;43(2):932-42. doi: 10.1093/nar/gku1353. Epub 2014 Dec 30.
The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n-2 and n-4/n-5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3'-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.
当复制性B族聚合酶将核苷酸正确添加到新链上时，核酸外切酶和聚合酶活性之间的平衡会促进DNA合成而非降解。错配会使平衡向核酸外切酶位点倾斜，而当错误的核苷酸被去除后，平衡又会重新偏向DNA合成。大多数B族DNA聚合酶都有一个延长的β-发夹环，这似乎对从核酸外切酶位点切换到聚合酶位点很重要，这个过程会影响DNA聚合酶的保真度。在这里，我们表明，尽管没有延长的β-发夹环，DNA聚合酶ε仍能以持续的方式在聚合酶位点和核酸外切酶位点之间切换。K967和R988是手掌和拇指结构域中的两个保守氨基酸，分别与引物链上n-2和n-4/n-5位置小沟中的碱基相互作用。DNA聚合酶ε依赖K967和R988来稳定聚合酶位点内DNA的3'末端，并依赖R988在核酸外切酶位点和聚合酶位点之间持续切换。基于与DNA聚合酶δ的结构比对，我们提出与R988对应的精氨酸在其他B族聚合酶中可能具有类似功能。
