DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase

Abstract

Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.adaptation | separase | securin | cell cycle arrest I n the presence of a single unrepairable DNA double-strand break (DSB), Saccharomyces cerevisiae arrest is due to Mec1 checkpoint kinase-dependent cell cycle arrest, but cells eventually escape from the arrest and reenter the cell cycle in a process termed adaptation (1-6). Arrest before adaptation typically lasts 12-15 h, a time equivalent to five to six normal cell cycles. Adaptation is accompanied by the loss of checkpoint-induced hyperphosphorylation of checkpoint kinases Rad53 and Chk1 and the loss of association with damaged DNA of the Mec1 (ATR) kinaseassociated Ddc2 (ATRIP), despite the persistence of DNA damage (7,8). Several proteins are required for adaptation, including those with known roles in DNA repair (Yku70, Yku80, Rdh54/ Tid1, Rad51, Srs2, Sae2, Fun30, and Sgs1), as well as proteins that are required to turn the checkpoint off (the PP2C phosphatases Ptc2 and Ptc3 and casein kinase II) and the Polo kinase Cdc5, which plays several roles in regulating mitosis (5,6,(9)(10)(11)(12)(13)(14).When DSB repair is relatively rapid, as during HO endonuclease-induced mating-type (MAT) switching, there is no cell cycle arrest or Rad53 phosphorylation (7), although Mec1 and Tel1 kinases rapidly phosphorylate histone H2A (15, 16). However, when DSB repair is slower, e.g., during ectopic homologous recombination or long-distance single-strand annealing, the DNA damage checkpoint is fully activated and G2/M arrest is maintained for several hours until repair is effected and cells reenter the cell cycle in a process termed recovery (12, 17). Recovery is an active process that requires the functions of the Srs2 helicase and also the casein kinase II and PP2C phosphatases to dephosphorylate checkpoint kinases (11-13). None of the other adaptation mutants tested (yku70Δ, rdh54Δ, rad51Δ, sae2Δ, cdc5-ad, and fun30Δ) is deficient in recovery (12, 14) although a number of double mutants are recovery defective (3). Analysis of checkpoint recovery in human cells has shown a requirement ...

Deacetylase Rpd3 Facilitates Checkpoint Adaptation by Preventing Rad53 Overactivation

“…Hyperactivation of the cytoplasm to vacuole targeting (CVT) autophagy pathway induces permanent G 2 /M arrest of cells with an unrepairable DSB. In this case, Rad53 overactivation was not observed (19).…”

Potential function for the Huntingtin protein as a scaffold for selective autophagy

“…Autophagy proteins can regulate cell cycle progression, mitosis, and selective midbody ring autophagic clearance (46)(47)(48); therefore, HTT activity at the mitotic spindle may reflect its function as part of the autophagic machinery. We also find that HTT knockout in the mouse CNS causes accumulation of p62-and ubiquitincontaining aggregates, which may reflect a loss of aggrephagy, the selective autophagic disposal of protein oligomers and aggregates (49).…”

Autophagy and genomic integrity

“…This arrest persists even when phosphorylation of the checkpoint kinase Rad53 is reduced, but is overcome when autophagy is blocked or vacuolar proteolysis is inhibited, suggesting that autophagy is fundamental for DNA damage-induced anaphase arrest, thus avoiding improper chromosome segregation. 44 Interestingly, elimination of the mid-body, which is involved in the final stages of cytokinesis, by autophagy was also shown to influence the tumorigenic potential of cancer cells. 45 Indeed, autophagy-defective cells exhibited nuclear morphometric alterations, centrosome abnormalities and increased chromosome number under normal culture conditions.…”