While PIF1 deletion rescues the lethality of Dna2 depletion, RAD9 ablation relieves the first cell cycle arrest causing genotoxicity after few cell divisions.
Slow fork speed attenuates DDR in Dna2 deprived cells.
While PIF1 deletion rescues cell lethality owing to Dna2 depletion, RAD9 ablation only relieves the first cell cycle block induced by the absence of Dna2, leading to Pif1-dependent ribosomal DNA (r DNA) chromosome fragmentation after few cell divisions.
Slowing down replication fork speed by hydroxyurea (HU) treatment or by lowering the temperature attenuates the DDR response and the first cell cycle arrest in cells deprived of Dna2 and suppresses the lethality of dna2 rad9 cells.
Okazaki fragment maturation depends on the strand displacement activity of Pol δ, which peels off the 5’end of the previous Okazaki fragment, Rad27/FEN1, which cuts the resulting DNA flap, and DNA nick ligation mediated by the DNA ligase I. USA 98, 5122–5127 (2001)." href="/articles/s41467-018-07378-5#ref-CR8" id="ref-link-section-d21795e414", contributes to the displacement of a fraction of 5’ ends of Okazaki fragments, forming long ss DNA tails that are cleaved, sequentially, by Dna2 and Fen1, through a secondary pathway of Okazaki fragment maturation known as alternative pathway of Okazaki fragment processing (APO).
a–d Dna2 was depleted in G1 in the indicated strains and cells were released into unperturbed S phase (a, b), S phase in the presence of a low dose of HU (c) or at a low temperature (d).
a, c Non-depleted cells were kept in parallel as control.
The data presented in this work suggest that the essential role of Dna2 during an unperturbed S phase is to counteract the formation of Pif1-and fork speed-dependent DNA flaps to prevent DDR hyper-activation and chromosome fragmentation.
We generated the conditional lethal Tc-dna2-AID allele (CL-dna2) to study the essential function of Dna2 (Fig. CL-dna2 mutants, experiencing one round of DNA synthesis under restrictive conditions, completed S phase with kinetics similar to control cells but arrested with a 2C DNA content, phosphorylated DNA polymerase alpha B subunit, a marker of G2 arrest, and hyper-phosphorylated Rad53, indicative of DDR activation (Fig. Preventing mitosis by nocodazole addiction did not influence DDR activation, while Dna2 depletion following S-phase completion precluded Rad53 activation (Supplementary Fig. Thus, DDR activation depends on chromosome replication but arises after S-phase completion.