Faithful DNA replication is required for transmission of the genetic material across generations. The basic mechanisms underlying this process are shared among all organisms: progressive unwinding of the long double-stranded DNA; synthesis of RNA primers; and synthesis of a new DNA chain. These activities are invariably performed by a multi-component machine called the replisome. A detailed description of this molecular machine has been achieved in prokaryotes and phages, with the replication processes in eukaryotes being comparatively less known. However, recent breakthroughs in the in vitro reconstitution of eukaryotic replisomes have resulted in valuable insight into their functions and mechanisms. In conjunction with the developments in eukaryotic replication, an emerging overall view of replisomes as dynamic protein ensembles is coming into fruition. The purpose of this review is to provide an overview of the recent insights into the dynamic nature of the bacterial replisome, revealed through single-molecule techniques, and to describe some aspects of the eukaryotic replisome under this framework. We primarily focus on Escherichia coli and Saccharomyces cerevisiae (budding yeast), since a significant amount of literature is available for these two model organisms. We end with a description of the methods of live-cell fluorescence microscopy for the characterization of replisome dynamics.
An ongoing mission for biologists is to probe the molecular nature of cellular processes within live cells. Although much of what we have discovered during the molecular biology revolution of the last 50 years has been achieved by exploiting bacteria as ‘bags of DNA and proteins’, relatively little has been learnt about how they organize their life processes within cells. The mistaken perception of bacteria cells as unstructured systems arose partly because of the difficulty of performing studies by light microscopy due to their small size (many of them having cell lengths a few times bigger than the wavelength of visible light). With the opportunities provided by a range of new fluorophores and by new microscopic techniques, a revolution in bacterial cell biology is revealing unimagined organization in the bacterial cell. We review the development and exploitation of new visualization methods and reagents and show how they are contributing to the understanding of bacterial structure, chromosome organization, DNA metabolism and their relationship to the cell cycle.