Department of Molecular Biology, Comenius University, Mlynská dolina B-2, 842 15, Bratislava 4, Slovak Republic.
Mol Biotechnol. 2014 Jan;56(1):1-11. doi: 10.1007/s12033-013-9680-6.
In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.
在先前从醋杆菌 CCM3610 菌株中分离的 pAG20 质粒中，Rep20 蛋白被鉴定为主要的复制起始因子。pAG20 质粒的原点定位于 rep20 基因附近，包含两个 21 个核苷酸长的倒位重复序列、两个 13 个核苷酸长的直接重复序列和一个 DnaA 结合位点。电泳迁移率变动分析和非放射性片段分析证实 Rep20 蛋白与两个直接重复序列（5'-TCCAAATTTGGAT'-3'）相互作用，并且通过突变验证了其在质粒复制过程中的必要性。尽管在复制起始过程中通常会发生宿主细胞大肠杆菌中的 DnaA 蛋白与质粒编码的复制起始因子的结合，但无法验证这种关联，Rep20 能够形成二聚体结构，通过这种结构它可以结合 rep20 基因的序列并自我调节其自身的表达。Rep20 蛋白的靶向突变揭示了第三α-螺旋和 63Lys 的重要性，特别是在 DNA 结合过程中。第二个紧密相邻的β-折叠也参与了这个过程，其中 52Asn 起着重要作用。
