Regulation of Sister Chromosome Cohesion by the Replication Fork Tracking Protein SeqA

Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.

与真核生物的染色体黏连（chromosome cohesion）机制类似，大肠杆菌（E. coli）中新复制的DNA在分离形成子代类核（nucleoid）之前，会通过姐妹链间连接（inter-sister linkages）维持结合状态。两种情形下的初始结合过程均由DNA连环（DNA catenation）介导：复制过程诱导产生的正超螺旋会在复制叉（replication fork）后方扩散，使新合成的双链DNA彼此相互缠绕。在真核生物中，功能已被充分阐明的黏连蛋白复合物（cohesin complex）会延缓由II型拓扑异构酶（Type-II topoisomerase）催化的姐妹染色体分离过程，但目前学界对大肠杆菌的黏连调控机制仍不明确。本研究报道，丰度较高的复制叉追踪蛋白SeqA是黏连过程的强效正向调控因子，也是在「snap」位点（snap loci）观察到的黏连现象显著延长的原因。上位性分析（epistasis analysis）显示，SeqA可通过拮抗拓扑异构酶IV（Topo IV）介导的姐妹染色体解离过程稳定黏连，此外还可能通过直接桥接机制发挥作用。我们证实，大肠杆菌染色体各区域的黏连程度存在差异，这源于SeqA的结合水平差异：其中复制起点oriC（oriC）与「snap」位点结合的SeqA比例显著更高。我们提出，SeqA结合会形成可抵抗Topo IV切割的松散双链间连接结构。最后，通过对Topo IV或SeqA进行基因操作以降低黏连水平后，姐妹位点分离过程显著减慢，类核分离效果也大幅受损，这表明黏连在染色体分离（chromosome segregation）过程中发挥着关键作用。