| Research Abstract |
SeqA protein binds preferentially hemimethylated GATC sequences, and regulates negatively initiation of chromosome replication in E. coli. We demonstrated that purified SeqA inhibits binding of initiation protein DnaA to hemimethylated oriC DNA. In newborn cells, two MukB-GFP fluorescent foci are located at 1/4 and 3/4 cellular positions and one SeqA focus is located at the mid-cell position. Subsequently, a SeqA focus is divided into two and those migrate to the cell quarter positions. Beta-subunit (DnaN, DNA sliding clump) of DNA polymerase III holoenzyme is localized in cytosolic spaces at the cell poles in non-replicating cells. However, when chromosome replication is initiated, the subunit is recruited into nucleoid and forms a cluster at mid-cell. A beta-subunit cluster is separated into two that are located at the cell quarter positions, when two SeqA clusters are located at the cell quarter positions. These results support our 'translocating replication factories' model, in which paired replication apparatuses that act for bi-directional replication are first located closely with each other at mid-cell, and then migrate to the cell quarter positions. We found that sister chromosomes are cohesive with each other after replication and the cohesion is released in late period of 41 chromosome replication. Cohesion of sister chromosomes was not observed in MukB null mutant cells, indicating that MukB participates in sister chromosome cohesion. We analyzed localization of nascent DNA segments that were pulse-labeled with 5-bromodeoxyuridine and found that MukB is essential for cohesion of two replication forks and also for proper localization of forks.
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