A solution to release twisted DNA during chromosome replication by coupled DNA polymerases

Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands(1). However, coupled replication presents a largely unrecognized topological problem. Since DNA polymerase must travel a helical path during synthesis, the physical connection between leading and lagging strand polymerases causes the daughter strands to entwine, or produces extensive buildup of negative supercoils in the newly synthesized DNA(2–4). How DNA polymerases maintain their connection during coupled replication despite these topological challenges is a mystery. Here, we examine the dynamics of the E. coli replisome, by ensemble and single-molecule methods that may solve this topological problem independent of topoisomerases. We find that the lagging strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome due to its contact with the leading-strand polymerase. This behavior, referred to as “signal release”, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments(5–7). However, we observe that signal release is independent of primase and does not appear to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.