Dataset Information

Phosphoproteomic Analysis of Mec1/ATR Signaling in Response to Different Manners of Kinase Activation

ABSTRACT: The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we employed a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of “free” Mec1 Activation Domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator-proteins, “free” MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1’s role in genome maintenance is separable from a previously unappreciated pro-replicative function. Both Mec1’s functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Michael Lanz

LAB HEAD: Marcus B Smolka

PROVIDER: PXD009734 | Pride | 2019-01-14

REPOSITORIES: Pride

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Publications

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

Lanz Michael Charles MC Oberly Susannah S Sanford Ethan James EJ Sharma Sushma S Chabes Andrei A Smolka Marcus Bustamante MB

Genes & development 20180613 11-12

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic-phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 ac ...[more]

PMID: 29899143

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Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.

Project description:Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. 30 total samples: (6 samples - BrdU- HU arrest 45min with 2 replicates, strains: WT, rif1 delta, pfa4 delta) (12 samples -S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: WT, rif1 delta, mec1_100) (12 samples - S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: sml1 delta, sml1 delta rif1 delta, sml1 delta mec1 delta)

Dataset Information

Phosphoproteomic Analysis of Mec1/ATR Signaling in Response to Different Manners of Kinase Activation

Publications

Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling.

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