A study of boundary regions of the Ter domain, which is organized at the replication terminus region of E. coli chromosome 大腸菌染色体の複製終結領域で構成されるTerドメインの境界領域に関する研究

博士論文

&nbsp;&nbsp;A circular chromosome of prokaryote has a single replication origin, oriC, at which bidirectional replication is initiated. Bidirectionally progressive replication forks meet at the opposite chromosomal region of oriC and the replication terminates. In general, this chromosomal region has been referred to the replication terminus. The large chromosomal region including the replication terminus is organized as a domain. It is called the Ter domain. In addition to the main function of the replication terminus, this chromosomal region plays a crucial role for physical separation of replicated daughter chromosomes. Recombination between circular daughter chromosomes can produce a jointed molecule of them, or dimer. The dimer of daughter chromosomes cannot physically segregate to each daughter cell. A mechanism of site-specific recombination is provided to circular bacterial chromosomes to resolve the dimer to monomers. In E.coli, an unique sequence for the dimer resolution (dif) is located at the center of the replication terminus. Interestingly, chromosomal position of dif is on just the opposite site of oriC and function of the dimer resolution. When dif is translocated out of the replication terminus, chromosomal dimers are accumulated at high frequently. To reveal positional effect of dif, chromosome arrangement of the terminus region has been studied using deletion mutants in the replication terminus. In consequence, it is thought that organization of the replication terminus contributes for effective function of the dimer resolution at dif.<br/>&nbsp;&nbsp;In addition, it is known that a membrane protein FtsK can help to resolve dimers. FtsK is integrated into inner membrane only at septum when cells divide. Thus, the chromosomal dimer can be effectively resolved according to the cell division cycle. An enzymatic function of FtsK is known as DNA translocase, which directionally pulls a DNA strand depending of specific sequences (KOPS). Consensus sequences of KOPS are scattered at the entire chromosome. They are not at random, but polarized distribution in the E.coli genome. KOPS is symmetrically distributed around the axis between oriC and dif on the E.coli circular chromosome so that the dif sites tend to be stayed at the middle of cells at which FtsK is positioned. Recently, it was found that the replication terminus is organized as folded structure by a DNA binding protein, MatP. The binding sites of MatP (matS) are distributed only within the replication terminus. Depending on growth conditions, cells with a mutation of matP are defective in chromosome segregation. However, the biological function of the MatP-matS DNA binding system is not clear. Thus, a complex system is involved in accurate segregation of circular chromosomes at the replication terminus. Probably the unique chromosomal configuration at the replication terminus promotes physical separation of daughter chromosomes. How and what chromosomal configuration of the replication terminus can contribute for chromosome segregation? In this study, a series of experiments using inversion mutants between the oriC and dif were carried out to investigate configuration of the terminus region for proper chromosome segregation.<br/>&nbsp;&nbsp;The analysis of chromosomes using inversion mutations, as well as deletion analysis of chromosome, is helpful to consider effects of chromosomal configuration on accurate chromosome segregation. In fact, it has been known that the inversion between two chromosomal positions, the 84.054 min locus near the oriC (84.6 min) and the 33.092 min locus near the dif (34.6 min), Inv(84.054-33.092), causes a defect in chromosome segregation so that anucleate cells are produced at high frequency (29% of total cells). It seems likely that the inversion, Inv(84.054-33.092), splits the replication terminus into two parts. To confirm it, localization of MatP fused with fluorescent protein mCherry was observed. While one or two discrete foci were seen in the wild type, the number of MatP-mCherry foci increased in the Inv(84.054-33.092) mutant, indicating that the replication terminus was split. In contrast, Inv(84.054-22.483) mutation, in which one inversion position is far from dif as compared with the Inv(84.054-33.092) and entire the replication terminus was inverted without splitting, hardly affects chromosome segregation. These results suggest that distance of the inversion position from dif is of critical importance for accurate chromosome segregation and splitting of replication terminus compromise it.<br/>&nbsp;&nbsp;To know why the splitting of terminus in the Inv(84.054-33.092) causes a defect in accurate chromosome segregation, effect of the genetic system was examined. Either mutation of ftsK or matP was introduced into the inversion mutants and frequency of production of anucleate cells was tested. The ftsK mutant that lacks its ATPase activity for DNA translocation remarkably reduced production of anucleate cells in the Inv(84.054-33.092). The matP null mutation also remarkably suppressed production of anucleate cells. Thus, both FtsK and MatP were closely related to the deficiency of accurate chromosome segregation in Inv(84.054-33.092).<br/>&nbsp;&nbsp;The results of the inversion mutants also suggest existence of the core of the terminus region required for accurate chromosome segregation. To identify the core of the terminus region, frequencies of producing anucleate cells were measured in a nested series of inversion mutants. The frequencies of producing anucleate cells increased gradually between 27.859 min and 28.469 min, and steeply between 28.469 min and 28.473 min. Thus, it is concluded that the boundary is located between 28.469 min and 28.473 min (268 kb far from dif). In this boundary, matS4, a single binding sequence of MatP, is located. Similar analysis identified the other boundary at 4.9 min (248 kb) far from dif in the opposite direction, where matS21 is located. Therefore, the core of the terminus region is located between 27.859 min and 39.592 min (544 kb). This region is referred to cTer.<br/> &nbsp;&nbsp;The 544 kb cTer region is thought to form folded structure by binding to MatP and this structure or organization is required for accurate chromosome segregation. This region also has dif that plays a curial role for dimer resolution and the dimer resolution requires a specialized mechanism to promote the directional reaction. Perhaps the specialized mechanism might be the chromosomal organization that is composed of cTer and might contribute to accurate chromosome segregation. Further analysis of cTer will reveal an exact mechanism for coupling the chromosome segregation and cytokinesis at septum, because FtsK localizes at septum when cells divide and brings the dif sites to the middle of the cells.<br/>

総研大甲第1433号