Idling by DNA polymerase δ maintains a ligatable nick during lagging-strand DNA replication

Abstract

During each yeast cell cycle, ∼100,000 nicks are generated during lagging-strand DNA replication. Efficient nick processing during Okazaki fragment maturation requires the coordinated action of DNA polymerase δ (Pol δ) and the FLAP endonuclease FEN1. Misregulation of this process leads to the accumulation of double-stranded breaks and cell lethality. Our studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol δ by default displaces 2–3 nt of any downstream RNA or DNA it encounters. In the presence of FEN1, efficient nick translation ensues, whereby a mixture of mono- and small oligonucleotides are released. If FEN1 is absent or not optimally functional, the ability of Pol δ to back up via its 3′–5′-exonuclease activity, a process called idling, maintains the polymerase at a position that is ideal either for ligation (in case of a DNA–DNA nick) or for subsequent engagement by FEN1 (in case of a DNA–RNA nick). Consistent with the hypothesis that DNA polymerase ϵ is the leading-strand enzyme, we observed no idling by this enzyme and no cooperation with FEN1 for creating a ligatable nick.