Defining lagging‐strand polymerase dynamics in vivo

At the replication fork, the leading strand can be continuously synthesized in the direction of DNA unwinding. However, the lagging strand must be discontinuously synthesized via the repeated priming, extension, processing and ligation of Okazaki fragments. Lagging‐strand synthesis in eukaryotes requires the sequential action of two polymerases – Pol α/primase and Pol δ. We have previously found that the location of Okazaki fragment termini is determined by interaction between Pol δ and newly deposited nucleosomes on the replicated DNA, and that this interaction generates fragments whose size is quantized according to the chromatin repeat (1). However, although the length of Okazaki fragments appears to be broadly similar across diverse eukaryotes and the mechanism that fine‐tunes Okazaki fragment size is known, it is not clear how the overall range of fragment length is determined. Moreover, although it is clear that the association of the lagging‐strand polymerases with DNA must be dynamic, the stoichiometry and stability of lagging‐strand polymerase binding at active replication forks have not been extensively investigated in eukaryotes. By titrating the levels of wild‐type and mutant replicative polymerases in S. cerevisiae and analyzing newly synthesized DNA on the lagging strand, we investigate the dynamics of DNA polymerase association with the replisome, the mechanisms by which eukaryotic Okazaki fragment length is specified and maintained in vivo , and the consequences for genome integrity of perturbing this length. Data from these investigations, and their mechanistic implications, will be discussed Support or Funding Information R01 GM114340 (NIGMS)Searle Scholars Program