Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replicationin vivo

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 clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. 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.

Ligation of newly replicated DNA controls the timing of DNA mismatch repair

“…To test this, we fused the G2/M tag to CDC9 to generate a strain in which CDC9 expression was restricted to G2/M so that replicationassociated nicks persist throughout S phase (Figure 4A). Consistent with a recent report, 37 strains carrying the G2/M-CDC9 allele were viable and had increased levels of unligated Okazaki S3A-S3C). In contrast, a strain carrying the cdc9-FFAA mutant allele, which is expected to have reduced ligase efficiency but normal expression timing, did not show DNA-damage checkpoint activation or an accumulation of Sphase cells (Figures S3A-S3C) and had less pronounced and slightly larger Okazaki fragments than G2/M-CDC9 when cells were arrested in S or G2/M phase (Figure S2).…”

Okazaki fragment maturation: DNA flap dynamics for cell proliferation and survival

“…An important but not fully answered question is what each of these three nucleases contributes during OFM. To address this issue, a deep DNA sequencing approach was used to analyze Okazaki fragments in mutant yeast strains that were conditionally depleted of Rad27, Dna2, or Exo1, either individually or in combination [20]. Consistent with the results of previous yeast genetic studies, Rad27 was found to be responsible for processing the majority of the 5′ flaps.…”

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

“…However, it should be noted that inhibiting DDR or critical DNA repair enzymes interferes with Pol II pause release and gene expression [4][5][6]17,24,89 , suggesting a proactive role of DNA break and DDR in transcriptional activation. Moreover, it is not difficult to find the examples of purposed and functional DNA fragmentation and small nucleic acid production in DNA metabolisms such as DNA replication 90 , recombination 91 , and repair 92 . In the future, it will be important to understand the role of TOP2-mediated DNA break and the mechanisms of TOP2 regulation for gene activation.…”