The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADPribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.
Edited by Patrick SungUracil N-glycosylase 2 (UNG2), the nuclear isoform of UNG, catalyzes the removal of uracil or 5-fluorouracil lesions that accumulate in DNA following treatment with the anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine), a 5-fluorouracil metabolite. By repairing these DNA lesions before they can cause cell death, UNG2 promotes cancer cell survival and is therefore critically involved in tumor resistance to these agents. However, the mechanisms by which UNG2 is regulated remain unclear. Several phosphorylation sites within the N-terminal regulatory domain of UNG2 have been identified, although the effects of these modifications on UNG2 function have not been fully explored, nor have the identities of the kinases involved been determined. Here we show that glycogen synthase kinase 3 (GSK-3) interacts with and phosphorylates UNG2 at Thr 60 and that Thr 60 phosphorylation requires a Ser 64 priming phosphorylation event. We also show that mutating Thr 60 or Ser 64 to Ala increases the half-life of UNG2, reduces the rate of in vitro uracil excision, and slows UNG2 dissociation from chromatin after DNA replication. Using an UNG2-deficient ovarian cancer cell line that is hypersensitive to floxuridine, we show that GSK-3 phosphorylation facilitates UNG2-dependent repair of floxuridine-induced DNA lesions and promotes tumor cell survival following exposure to this agent. These data suggest that GSK-3 regulates UNG2 and promotes DNA damage repair.2 is a member of the uracil DNA glycosylase family of enzymes, which initiate base excision repair (BER) of uracil that results from deamination of cytosine or the misincorporation of uracil or other thymidine analogs during DNA replication (1-4). Importantly, UNG is the main glycosylase that removes uracil and 5-fluorouracil lesions that accumulate in DNA following treatment of cancers with the closely related fluoropyrimidine anticancer agents 5-fluorouracil and 5-fluorodeoxyuridine (floxuridine) (5-7). Consistent with this observation, we and others have shown previously that UNG protects cells from death induced by agents that cause incorporation of uracil or 5-fluorouracil during DNA replication, likely by removing these lesions before they can cause cell death (4, 6 -8).Mammalian cells express two isoforms of UNG: UNG1, a mitochondrial isoform, and UNG2, a nuclear isoform. Both isoforms have identical C-terminal catalytic domains but differ in the N-terminal regulatory domain because of differential promoter usage and alternative splicing (9). These isoforms are expressed highly during late G 1 /early S phase of the cell cycle and are reduced in late S phase through proteasome-mediated degradation (10, 11).UNG initiates BER by detecting and excising the damaged base, leaving an apyrimidinic site, which recruits an apurinic/ apyrimidinic endonuclease (12) to cleave the DNA backbone 5Ј to the damaged base. The single-stranded nick is then repaired through the subsequent activity of a DNA polymerase and DNA ligase. Although BER can be...
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