The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.
WMS - Cell and Development Biology, University of Warwick, Coventry, CV4 7AL, UK.
Sci Rep. 2018 Jan 19;8(1):1206. doi: 10.1038/s41598-017-18817-6.
Bacterial antibiotic resistance is often carried by circular DNA plasmids that are copied separately from the genomic DNA and can be passed to other bacteria, spreading the resistance. The chloramphenicol-resistance plasmid pC221 from Staphylococcus aureus is duplicated by a process called asymmetric rolling circle replication. It is not fully understood how the replication process is regulated but its initiation requires a plasmid-encoded protein called RepD that nicks one strand of the parent plasmid at the double-stranded origin of replication (oriD). Using magnetic tweezers to control the DNA linking number we found RepD nicking occurred only when DNA was negatively supercoiled and that binding of a non-nicking mutant (RepDY188F) stabilized secondary structure formation at oriD. Quenched-flow experiments showed the inverted complementary repeat sequence, ICRII, within oriD was most important for rapid nicking of intact plasmids. Our results show that cruciform formation at oriD is an important control for initiation of plasmid replication.
细菌对抗生素的耐药性通常由环状 DNA 质粒携带，这些质粒与基因组 DNA 分开复制，可以传递给其他细菌，从而传播耐药性。金黄色葡萄球菌的氯霉素耐药质粒 pC221 通过一种称为不对称滚环复制的过程进行复制。尽管人们并不完全了解复制过程是如何调控的，但它的起始需要一种质粒编码的蛋白 RepD，该蛋白在双链复制起点 (oriD) 处切割亲本质粒的一条链。我们使用磁镊来控制 DNA 的连接数，发现 RepD 切割仅发生在 DNA 负超螺旋时，并且非切割突变体 (RepDY188F) 的结合稳定了 oriD 处的二级结构形成。猝灭流实验表明，oriD 内的互补反向重复序列 ICRII 对完整质粒的快速切割最为重要。我们的结果表明，oriD 处的十字形形成是质粒复制起始的重要控制因素。
