Abstract
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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.