Recycling of single-stranded DNA-binding protein by the bacterial replisome
Lisanne M Spenkelink Jacob S Lewis Slobodan Jergic Zhi-Qiang Xu Andrew Robinson Nicholas E Dixon Antoine M van Oijen
Nucleic Acids Research, gkz090, https://doi.org/10.1093/nar/gkz090
Published: 15 February 2019
Article history
Received: 18 September 2018 Revision Received: 30 January 2019
Accepted: 09 February 2019
Abstract
Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.
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