Spenkelink, L. M. | bioRxiv Science

bioRxiv2019

Single-molecule imaging of eukaryotic replisomes reveals compositional plasticity

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol DNA polymerase activity in replication and show that Pol primase and the lagging-strand Pol {delta} can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

biophysics↗

bioRxiv2018

Recycling of single-stranded DNA-binding protein by the bacterial replisome.

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 visualise how an in vitro reconstituted E. 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 visualising SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant.

biophysics↗