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

• 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.

Nyckelord

DNA/*metabolism

DNA Helicases/metabolism

DNA Polymerase III/*metabolism

DNA Primers

DNA Replication

Exonucleases/metabolism

Flap Endonucleases/*metabolism

Oligonucleotides/metabolism

Saccharomyces cerevisiae/*enzymology/*genetics

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