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

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 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 proreplicative 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.

DNA damage kinase signaling: checkpoint and repair at 30 years.

From bacteria to mammalian cells, damaged DNA is sensed and targeted by DNA repair pathways. In eukaryotes, kinases play a central role in coordinating the DNA damage response. DNA damage signaling kinases were identified over two decades ago and linked to the cell cycle checkpoint concept proposed by Weinert and Hartwell in 1988. Connections between the DNA damage signaling kinases and DNA repair were scant at first, and the initial perception was that the importance of these kinases for genome integrity was largely an indirect effect of their roles in checkpoints, DNA replication, and transcription. As more substrates of DNA damage signaling kinases were identified, it became clear that they directly regulate a wide range of DNA repair factors. Here, we review our current understanding of DNA damage signaling kinases, delineating the key substrates in budding yeast and humans. We trace the progress of the field in the last 30 years and discuss our current understanding of the major substrate regulatory mechanisms involved in checkpoint responses and DNA repair.

MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity.

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

The Rad53CHK1/CHK2-Spt21NPAT and Tel1ATM axes couple glucose tolerance to histone dosage and subtelomeric silencing.

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.