The proliferation of all organisms depends on the coordination of enzymatic events within large multiprotein replisomes that duplicate chromosomes. Whereas the structure and function of many core replisome components have been clarified, the timing and order of molecular events during replication remains obscure. To better understand the replication mechanism, new methods must be developed that allow for the observation and characterization of short-lived states and dynamic events at single replication forks. Over the last decade, great progress has been made toward this goal with the development of novel DNA nanomanipulation and fluorescence imaging techniques allowing for the direct observation of replication-fork dynamics both reconstituted in vitro and in live cells. This article reviews these new single-molecule approaches and the revised understanding of replisome operation that has emerged.
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