Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo.

Malik Kahli,Duncan J Smith,Joseph S Osmundson,Rani Yeung

doi:10.1093/nar/gky1242

Abstract

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5′ flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5′ flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

Highlights

The synthesis of each Okazaki fragment requires several distinct enzymatic activities (Burgers, 2009)
We investigated Okazaki fragment processing in the absence of all combinations of the Okazaki fragment nucleases Rad27, Dna2 and Exo1 by carrying out a systematic in vivo analysis of Okazaki fragment size, ligatability and terminus location
Okazaki fragments are generated even in the absence of all three nucleases (Fig. 1), and that strand displacement synthesis by Pol δ is limited by the absence of nucleolytic Okazaki fragment cleavage (Figs. 2&3)

Summary

Introduction

The synthesis of each Okazaki fragment requires several distinct enzymatic activities (Burgers, 2009). Pol d synthesizes DNA to the 5’ end of the preceding fragment and continues beyond this point (Garg et al, 2004), generating a 5’ flap structure. Iterative rounds of extension, followed by flap cleavage or nick regeneration by the 3’-5’ exonuclease activity of Pol d (Garg et al, 2004), maintain a ligatable nick that persists until it is sealed by DNA ligase I, encoded by the CDC9 gene in S. cerevisiae (Johnston and Nasmyth, 1978). Nuclease cleavage during Okazaki fragment biogenesis represents an extremely abundant DNA transaction – one that must occur tens of thousands of times during each S-phase in S. cerevisiae and millions of times per human cell division

Methods

Results

Conclusion