Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.

Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex.

Macroautophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by the TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and macroautophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. Here, we show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate macroautophagy by dephosphorylating Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy and the cytoplasm-to-vacuole targeting pathway are inhibited, and the recruitment of the essential autophagy machinery to the phagophore assembly site is impaired. Expressing a genomic ATG13-8SA allele lacking key TORC1 phosphorylation sites partially bypasses the macroautophagy defect in ptc2Δ ptc3Δ strains. Moreover, Ptc2 and Ptc3 interact with the Atg1-Atg13 complex. Taken together, these results suggest that PP2C-type phosphatases promote macroautophagy by regulating the Atg1 complex.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2.

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells.

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint.

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2 and Rad53; however, after 15 hours both proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies.

Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.

Dephosphorylation of the Atg1 kinase complex by type 2C protein phosphatases.

In budding yeast, Atg1 kinase, together with Atg13 and Atg17, forms a complex that is essential for autophagy. Previous work showed that the Atg1 kinase complex is regulated extensively by phosphorylations. Our recent paper demonstrates that type 2C protein phosphatases Ptc2 and Ptc3 are involved in the dephosphorylation of Atg13 and Atg1 kinase to promote autophagy.

Mec1ATR Autophosphorylation and Ddc2ATRIP Phosphorylation Regulates DNA Damage Checkpoint Signaling.

In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1ATR-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint signaling and adapt despite the persistence of the DSB. We report that the adaptation involves the autophosphorylation of Mec1 at site S1964. A non-phosphorylatable mec1-S1964A mutant causes cells to arrest permanently in response to a single DSB without affecting the initial kinase activity of Mec1. Autophosphorylation of S1964 is dependent on Ddc1Rad9 and Dpb11TopBP1, and it correlates with the timing of adaptation. We also report that Mec1's binding partner, Ddc2ATRIP, is an inherently stable protein that is degraded specifically upon DNA damage. Ddc2 is regulated extensively through phosphorylation, which, in turn, regulates the localization of the Mec1-Ddc2 complex to DNA lesions. Taken together, these results suggest that checkpoint response is regulated through the autophosphorylation of Mec1 kinase and through the changes in Ddc2 abundance and phosphorylation.