Mechanisms of Chromosome Maintenance in Bacteria

我们的兴趣是了解DNA复制频率在细胞周期中是如何调整的。我们一直在使用质粒P1进行研究，其拷贝数控制机制似乎在细菌中很常见。其机制包括对引发剂蛋白合成的自体控制、通过二聚化使引发剂蛋白失活、通过分子伴侣将失活的二聚体转化为有活性的单体、引发剂结合到起始点的多个位点，以及通过结合的引发剂使起始点失活而对起始点进行配对。这些多种控制模式通过增加拷贝数少于平均拷贝数的细胞的复制速率和降低质粒过多的细胞的复制速率，有助于将质粒拷贝数维持在狭窄的范围内。质粒拷贝数的稳态研究为生物系统中如何调节波动提供了一个模型。

Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. We have been studying this using plasmid P1 whose mechanism of copy number control appears to be common in bacteria. The mechanism involves autogenous control on initiator protein synthesis, initiator protein inactivation by dimerization, conversion of inactive dimers to active monomers by molecular chaperones, initiator binding to multiple sites in the origin, and pairing of origins via the bound initiators that inactivates the origins. These multiple modes of control help to maintain plasmid copy number within narrow limits by increasing the rate of replication in cells that have fewer than average copy number and reducing the rate in cells that have too many plasmids. The study of homeostasis of plasmid copy number provides a model of how fluctuations can be adjusted in biological systems. Recent developments in genetic and fluorescent-based microscopic techniques have shown that the processes of chromosome replication and segregation are connected both in bacteria and in yeast. We have developed double-labeling techniques using easily distinguished fluorescent proteins to mark two chromosomal loci simultaneously in E. coli and analyze their segregation pattern. Our premise is that the locus that anchors daughter chromosomes to the cell poles will localize to a pole first, and thus qualify as a bacterial centromere. We are extending our studies to V. cholerae chromosomes. The genome of this bacterium is split into two chromosomes. Our interest is to identify the control elements of replication for the two chromosomes. This should help to understand how the chromosomes are maintained and whether their replication is under a check-point control that ensures complete replication of both the chromosomes before cell division. The location of the two chromosomes is being studied by the double-labeling technique described above.