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• W2967538289 abstract "Review8 August 2019free access DNA damage kinase signaling: checkpoint and repair at 30 years Michael Charles Lanz Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Diego Dibitetto Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Marcus Bustamante Smolka Corresponding Author [email protected] orcid.org/0000-0001-9952-2885 Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Michael Charles Lanz Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Diego Dibitetto Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Marcus Bustamante Smolka Corresponding Author [email protected] orcid.org/0000-0001-9952-2885 Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Search for more papers by this author Author Information Michael Charles Lanz1, Diego Dibitetto1 and Marcus Bustamante Smolka *,1 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA *Corresponding author. Tel: +1 607 2550274; E-mail: [email protected] EMBO J (2019)38:e101801https://doi.org/10.15252/embj.2019101801 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Glossary BER Base excision repair BIR Break-induced replication CDK Cyclin-dependent kinase DDK Dbf4-dependent kinase dNTP deoxyribonucleotide GCR Gross chromosomal rearrangement HR Homologous recombination ICL Interstrand crosslink IR Ionizing radiation MMR Mismatch repair NER Nucleotide excision repair NHEJ Non-homologous end joining PIKK PI3 kinase-related kinase RNR Ribonucleotide reductase SAC Spindle assembly checkpoint SDSA Synthesis-dependent strand annealing SSA Single-strand annealing Introduction From pathway to network In eukaryotes, kinases play a central role in the DNA damage response, from sensing DNA damage to regulating cellular processes. Work in yeast and mammalian systems in the 1990s identified an evolutionarily conserved set of DNA damage signaling kinases, including phosphatidylinositol 3′ kinase (PI3K)-related kinases (PIKKs) and PIKK-regulated downstream kinases. These kinases were found to be involved in cell cycle control (Allen et al, 1994; Carr, 1995; Morrow et al, 1995; Savitsky et al, 1995; Cimprich et al, 1996; Furnari et al, 1997; Peng et al, 1997; Sanchez et al, 1997) and were linked to the “cell cycle checkpoint” concept proposed by Weinert and Hartwell (Weinert & Hartwell, 1988; Hartwell & Weinert, 1989). Subsequent work in the late 1990s and early 2000s revealed how these kinases establish the checkpoint and control processes beyond the cell cycle, such as apoptosis, transcription, and DNA replication (Santocanale & Diffley, 1998; Sun et al, 1998; Zhou & Elledge, 2000). In 2007, the use of mass spectrometry (MS)-based proteomics allowed a more systematic analysis of the network of phosphorylation events triggered by DNA damage signaling kinases (Matsuoka et al, 2007; Smolka et al, 2007). As a result, the perception that DNA damage signaling kinases operate within a simple signaling pathway (Fig 1; the classical “linear” depiction of DNA damage signaling) evolved to a more comprehensive view in which DNA damage signaling kinases function in an elaborate signaling network comprised of hundreds of substrates. Figure 1. DNA damage signaling via PIKKs and checkpoint kinases in budding yeast and humansDNA damage signaling is initiated at DNA structures that form during DNA damage or replication stress, including single-strand DNA (ssDNA) and broken DNA ends. The apical PIKKs are recruited to these structures and become activated to initiate downstream signaling. Mec1/ATR is recruited to RPA-coated ssDNA, while Tel1/ATM and DNA-PKcs initially associate with DNA ends formed by double-strand breaks. Adaptor proteins are often required to mediate the transfer of phosphorylation from apical to downstream checkpoint kinases. Apical and downstream checkpoint kinases function coordinately to mediate cellular responses to DNA damage, either directly or through the regulation of additional downstream kinases. PIKKs also target an extensive network of substrates independently of downstream checkpoint kinases. Download figure Download PowerPoint From checkpoint to DNA repair After the discovery of DNA damage signaling kinases, mechanistic links between these kinases and the DNA repair machinery were virtually non-existent. It was not immediately appreciated that these kinases directly target and regulate the DNA repair machinery. Now that dozens of DNA repair proteins have been shown to be phosphorylated by DNA damage signaling kinases, there is little doubt that active and direct control of the DNA repair machinery is a core function of DNA damage signaling kinases. However, the precise mechanisms by which these kinases control the action of these substrates remain incompletely understood and represent a significant knowledge gap in the field. Here, we review our current understanding of the integrated action of DNA damage signaling kinases. We delineate the key substrates for checkpoint and DNA repair in budding yeast and highlight the potential parallels in humans. Based on the accumulated knowledge of the mechanisms of substrate regulation, we discuss how our understanding of the action of DNA damage signaling kinases in genome maintenance has evolved over the last 30 years. DNA damage signaling kinases DNA damage signaling kinases have been traditionally categorized as either apical or effector kinases. The apical PIKKs, or ATR, ATM, and DNA-PKcs in mammals and Mec1 and Tel1 in budding yeast (Fig 1; see Table 1 for gene name overview in model organisms), associate with DNA structures that form as byproducts of DNA damage or replication stress, including single-strand DNA (ssDNA) and broken DNA ends (Fig 1). In the canonical mode of action, Mec1/ATR is recruited to ssDNA, whereas Tel1/ATM and DNA-PKcs associate with the ends of double-strand DNA (dsDNA) breaks. The mechanism of activation for each of these kinases is different, but recruitment to damaged DNA is often a requirement (for details on the mechanism of how kinases associate with DNA structures, as well as co-factors involved, refer to the following reviews: Blackford & Jackson, 2017; Di Domenico et al, 2014; Saldivar et al, 2017; Zou, 2013). Activated apical kinases transfer stimulatory phosphorylation to the downstream checkpoint kinases (Rad53, Chk1, and Dun1 in yeast; CHK2 and CHK1 in mammals), which catalyze phosphorylation events that mediate cellular responses to DNA damage as part of the canonical DNA damage checkpoint (Fig 1). Whereas Rad53 mediates nearly all checkpoint-related functions in budding yeast, with Chk1 playing only a minor role (Sanchez et al, 1999), a more balanced division of labor exists for CHK1 and CHK2 in humans. Human CHK2 shares sequence and structural similarities with yeast Rad53, including an FHA domain (Matsuoka et al, 1998), but the functional similarities are limited to the DSB signaling response. Human CHK1 and yeast Rad53 play a crucial role in the replication stress response, but they share little or no sequence/structural similarity. Table 1. Key protein names in budding yeast, fission yeast, and humans Budding yeast Fission yeast Humans Apical PIKK kinase Mec1 Rad3 ATR Tel1 Tel1 ATM DNA-PKcs Downstream checkpoint kinase Rad53 Cds1 CHK2/CHK1aa Human CHK1 is a functional analog of yeast Rad53. Chk1 Chk1 CHK1? MRN complex Mre11 Mre11 MRE11 Rad50 Rad50 RAD50 Xrs2 Nbs1 NBS1 ATR cofactor Lcd1 (Ddc2) Rad26 ATRIP 9-1-1 complex Ddc1 Rad9 RAD9 Mec3 Hus1 HUS1 Rad17 Rad1 RAD1 ATR activators Dpb11 Rad4/Cut5 TOPBP1 Dna2 Ddc1 ETAA1 Adaptors Rad9 Crb2 MDC1/53BP1 Mrc1 Mrc1 CLASPIN a Human CHK1 is a functional analog of yeast Rad53. Adaptor proteins as substrates for downstream signaling activation In budding yeast, the transfer of phosphorylation from apical to downstream checkpoint kinases requires the checkpoint adaptor proteins Rad9 and Mrc1, which recruit the downstream checkpoint kinases in proximity to the apical kinase. Mec1 and Tel1 promote the recruitment of the Rad9 adaptor by phosphorylating lesion-proximal substrates, such as histone H2A (Downs et al, 2000) and the 9-1-1 complex (Paciotti et al, 1998), which are then recognized directly or indirectly by Rad9 (Fig 2; Toh et al, 2006; Hammet et al, 2007; Pfander & Diffley, 2011). Once recruited, Rad9 is phosphorylated by Mec1 or Tel1 (Emili, 1998; Vialard et al, 1998), which promotes its oligomerization (Soulier & Lowndes, 1999; Usui et al, 2009) and further stabilization on DNA (Naiki et al, 2004). Mec1- and Tel1-mediated phosphorylation of Rad9 creates docking sites for the recruitment of the downstream effector kinase Rad53 (Fig 2; Gilbert et al, 2001; Schwartz et al, 2002; Sun et al, 1998), which, upon recruitment to Rad9, is phosphorylated and activated by Mec1 or Tel1 (Sanchez et al, 1996; Sun et al, 1996). Chk1 also relies on Rad9 for its activation by Mec1 (Blankley & Lydall, 2004); however, unlike Rad53, the phosphorylation events that facilitate the recruitment of Chk1 to Rad9 are catalyzed by cyclin-dependent kinase (CDK; Fig 2; Abreu et al, 2013). Rad53 can also be activated via the Mrc1 adaptor (Alcasabas et al, 2001; Osborn & Elledge, 2003). Mrc1, being an intrinsic component of the replisome, is already “on-site” for mediating activation, obviating the need for a devoted recruitment mechanism, as is the case with Rad9. In fact, Mrc1 mediates a more rapid response to replication stress than Rad9-dependent DNA damage signaling (Pardo et al, 2017; Bacal et al, 2018). Similar to Rad9 and Mrc1, Sgs1 has been proposed to mediate Rad53 recruitment in a manner that depends upon its phosphorylation by Mec1 (Hegnauer et al, 2012). Mec1 and Tel1 also facilitate DNA damage signaling activation and propagation by recruiting chromatin modifiers and remodelers near sites of DNA damage (van Attikum et al, 2004; Downs et al, 2004; Morrison et al, 2004, 2007), which may help de-condense chromatin in a way that permits adaptor assembly. Figure 2. Recruitment of DNA damage signaling kinases and adaptor proteins to DNA lesions: conserved features between budding yeast and humansPhosphorylation and adaptor proteins play a key role in the recruitment of downstream checkpoint kinases. The colored ovals indicate phosphorylation events mediated by DNA damage signaling kinases (see kinase key). The orange lines indicate protein–protein interactions promoted by the indicated phosphorylation events (also methylation (me) or ubiquitylation (Ub)). Activation of the downstream checkpoint kinases by the apical PIKK kinases requires adaptor proteins (outlined in green). In most cases, these adaptor proteins act as scaffolds to directly bind to and recruit the downstream checkpoint kinase. The model, mostly based on extensive work in yeast, posits that the recruitment of the downstream checkpoint kinase to the proximity of the apical PIKK kinase enables the phosphorylation and activation of the downstream checkpoint kinase. In addition to activating the downstream checkpoint kinase, phosphorylation events mediated by the apical PIKK kinases are critical for scaffold assembly, often promoting protein–protein interactions. Accordingly, a conserved feature of several adaptor proteins in budding yeast and humans is the presence of protein domains responsible for binding phosphorylated proteins (FHA and BRCT domains). Notably, other kinases such as CDK and CK2 also catalyze phosphorylation events involved in adaptor recruitment, although these events are often not induced by DNA damage. For DNA-PKcs, while this kinase has been implicated in the phosphorylation of H2AX and 53BP1, it does not seem to be involved in CHK2 phosphorylation. Download figure Download PowerPoint Similar as in yeast, vertebrate ATM and ATR utilize checkpoint adaptor proteins to mediate their transfer of phosphorylation to the checkpoint effector kinases. ATR primarily relies on Claspin, the homolog of yeast Mrc1, to mediate the activation of CHK1 (Kumagai & Dunphy, 2000). Like Mrc1, Claspin associates with the replisome (Lee et al, 2003) and recruits CHK1 upon exposure to replication stress. Also similar to yeast, the Claspin–CHK1 interaction depends on ATR activity (Kumagai & Dunphy, 2003; Lindsey-Boltz et al, 2009). While it is currently unknown whether mammalian ATR directly phosphorylates Claspin, Xenopus ATR has been shown to directly phosphorylate Claspin at threonines 817 and 819, which are critical for CHK1 recruitment (Fig 2; the uncertainty of ATR's direct phosphorylation of Claspin in humans is denoted by “?”; Yoo et al, 2006; in-depth discussion of the similarities and differences between Claspin orthologs can be found here: Smits et al, 2019). Once bound to CHK1, Claspin is thought to both stabilize CHK1 and tether it in proximity to ATR, allowing for extensive phosphorylation and full activation of the effector kinase (Liu et al, 2006). While ATR utilizes Claspin to facilitate its phosphorylation of CHK1, the phosphorylation of many other ATR substrates does not require Claspin, suggesting that, like in yeast, the adaptor proteins are primarily responsible for facilitating Mec1/ATR's phosphorylation of the effector kinases and are not required for the phosphorylation of other substrates (like many of those depicted below in Fig 4). Two mammalian adaptors have been linked to the ATM–CHK2 signaling axis: MDC1 and 53BP1. Despite extensive research on these proteins, it remains unclear precisely how they function in transducing ATM signaling toward CHK2 activation. While 53BP1 is the functional ortholog of yeast Rad9, MDC1 also shares functional similarities with Rad9 in the context of the DNA damage signaling response. Similar to Rad9, MDC1 possesses BRCT domains that directly bind to phosphorylated histone H2AX (Fig 2; γH2AX; analogous to histone H2A phosphorylated at the C-terminal SQ site in yeast; Stucki et al, 2005). Once associated with γH2AX at DNA breaks, MDC1 is phosphorylated by ATM and contributes to CHK2 activation (Goldberg et al, 2003; Peng & Chen, 2003; Stewart et al, 2003; Wu et al, 2008). MDC1 has also been shown to be important for CHK1 activation through its interaction with the TOPBP1 scaffold (Wang et al, 2011; Leung et al, 2013), which may functionally resemble the Rad9–Dpb11 interaction in budding yeast (Fig 2). ATM promotes the recruitment of 53BP1 through two distinct mechanisms. Similar to yeast Rad9, 53BP1 recognizes γH2AX through its C-terminal pair of BRCT domains (Fig 2), an interaction which has been controversial, but has recently gained additional support (Baldock et al, 2015; Kleiner et al, 2015). Nonetheless, the prominent mechanism of 53BP1 recruitment to DNA breaks involves ATM- and γH2AX-mediated recruitment of MDC1, which becomes phosphorylated by ATM and recruits the E3 ubiquitin ligases RNF8 and RNF168, leading to ubiquitylation of H2A that is recognized directly by 53BP1 (Fig 2; for a detailed review, Hustedt & Durocher, 2016). Notably, this ubiquitylation-dependent recruiting mechanism is absent in budding yeast. Consistent with an adaptor function for 53BP1, it interacts with CHK2, and the loss of 53BP1 results in reduced ATM-mediated phosphorylation of CHK2 in response to low doses of ionizing radiation (IR; Wang et al, 2002). However, 53BP1 appears to regulate activation of the checkpoint through a more complex mechanism, as the physical interaction between CHK2 and 53BP1 rapidly decreases upon IR radiation rather than becoming stabilized (Wang et al, 2002), in contrast to the Rad9–Rad53 interaction, which increases after DNA damage in budding yeast. Both 53BP1 and Rad9 play a key role in the control of DNA end resection, highlighting the connection and coordination between checkpoint signaling and the regulation of DNA repair (discussed in detail later in this review; Lazzaro et al, 2008; Bunting et al, 2010; Chapman et al, 2013; Zimmermann et al, 2013; Ferrari et al, 2015; Liu et al, 2017). In both cases, phosphorylation of Rad9/53BP1 by Mec1 or Tel1 in yeast or ATM in humans is essential for suppressing DNA end resection (Bothmer et al, 2011; Ferrari et al, 2015), and it is possible that 53BP1's function in preventing resection contributes to the stabilization of ATM at breaks, which may indirectly promote ATM–CHK2 signaling. Post-recruitment events in downstream checkpoint kinase activation Recruitment to sites of DNA lesions via checkpoint adaptors enables the downstream checkpoint kinases to be directly phosphorylated by the upstream PIKKs, triggering initial kinase activation and subsequent autophosphorylation for further kinase activation. In the case of Rad53, for example, initial phosphorylation by Mec1 or Tel1 promotes its kinase activity by interfering with a kinase auto-inhibitory domain (Fiorani et al, 2008), which then stimulates Rad53 to phosphorylate other Rad9-bound Rad53 molecules (Jia-Lin Ma & Stern, 2008). Such trans-autophosphorylation events contribute to dissociate Rad53 from Rad9 and prevent further Rad9 oligomerization (Usui et al, 2009). Notably, overexpression of Rad53 in bacteria cells, which lack PIKKs and checkpoint adaptors, results in hyper-activated Rad53 (Gilbert et al, 2001). This finding supports a model whereby increased local concentration of Rad53 is enough for activation, with adaptors building increased local concentration at the site of lesions and PIKKs facilitating the initial trigger by reducing the minimal concentration threshold required for activation. In mammals, the apical kinases ATR and ATM drive the key events leading to activation of CHK1 and CHK2, respectively. ATR- and ATM-mediated phosphorylation not only recruits CHK1 and CHK2 to sites of DNA lesions, but also directly phosphorylates these downstream kinases to promote their activation. Like in yeast, these priming phosphorylation events are mainly required to relieve inhibitory domains or to drive monomer-to-oligomer kinase transition (reviewed in Bartek & Lukas, 2003). ATM-mediated phosphorylation of CHK2 at threonine 68, an established marker of CHK2 activation, allows for the dimerization of two inactive CHK2 monomers and for their subsequent trans- and cis-phosphorylation (Ahn et al, 2000; Xu et al, 2002; Schwarz et al, 2003). CHK2 dimerization is a transient state, since the multiple trans- and cis-autophosphorylation events promote rapid dimer dissociation, leading to full active monomers (Ahn & Prives, 2002; Xu et al, 2002; Cai et al, 2009). Similar to CHK2, CHK1 activation is a multistep process that requires ATR phosphorylation at serine 317 and serine 345; however, unlike Rad53 or CHK2, CHK1 activation does not appear to involve dimerization or oligomerization (Liu et al, 2000; Zhao & Piwnica-Worms, 2001). Substrates mediating the core DNA damage signaling responses Once activated, DNA damage signaling kinases mediate hallmark responses that include the arrest of the cell cycle, inhibition of origin firing, protection and restart of stalled replication forks, induction of a transcriptional response, initiation of apoptosis, and control of dNTP levels. More recent work has demonstrated that the DNA damage signaling kinases also regulate a range of other processes, such as autophagy, gene gating, chromosome mobility, transcription–replication conflicts, and many more whose mechanistic connections to DNA damage signaling and degrees of conservation across eukaryotes remain less clear. Here, we focus on a select set of core conserved functions of the DNA damage signaling kinases with defined substrates, delineating the parallels between budding yeast and humans (Fig 3). Figure 3. Yeast-to-human parallels in core checkpoint responses mediated by DNA damage signalingSubstrate map highlighting the phosphorylation events involved in core DNA damage signaling responses in yeast and humans (see text for detailed discussion of each substrate). Conserved or functionally analogous phosphorylation events are positioned parallel to each another. The colored ovals indicate phosphorylation events mediated by DNA damage signaling kinases (see “kinase-dependency” key). The arrows or lines that emanate from the colored ovals represent the role phosphorylation plays in regulating that protein (see “role of phosphorylation” key). Question marks indicate uncertainty, either in the functionality of the phosphorylation event or in the identity of the kinase or substrate. Arrows that impinge on CDK demonstrate how DNA damage signaling can indirectly inhibit CDK activity. Download figure Download PowerPoint Cell cycle control The most classical and widely known function of DNA damage signaling kinases is the imposition of a cell cycle arrest that prevents entry into mitosis. Exactly how this arrest is imposed in budding yeast remains elusive. The paradigm for how cell cycle arrest occurs comes from a series of works conducted in fission yeast and metazoans in 1997 (Furnari et al, 1997; Peng et al, 1997; Sanchez et al, 1997; Weinert, 1997), which revealed that DNA damage signaling inhibits CDC25, a phosphatase that removes inhibitory phosphorylation at a key tyrosine residue in mitotic-CDK (M-CDK; Fig 3). In addition, DNA damage signaling kinases stimulate WEE1 (O'Connell et al, 1997; Boddy et al, 1998; Lee et al, 2001), a kinase responsible for phosphorylating the same inhibitory tyrosine site (Fig 3; Mueller et al, 1995). While DNA damage signaling in budding yeast does not appear to impinge upon the Cdc25 phosphatase homolog Mih1, the budding yeast kinase Swe1 (the WEE1 homolog) is regulated similar to its counterpart in higher eukaryotes and fission yeast. Swe1 is likely phosphorylated and activated by DNA damage signaling, which results in inhibition of M-CDK (Fig 3; Edenberg et al, 2015; Palou et al, 2015). DNA damage signaling in budding yeast is also able to suppress M-CDK activity through additional redundant mechanisms that remain unclear (Palou et al, 2015). In addition to inhibiting M-CDK-dependent activation of the anaphase-promoting complex (APC/C), DNA damage signaling in budding yeast more directly inhibits the onset of anaphase through Chk1, which phosphorylates and stabilizes the anaphase-inhibiting Pds1/securin protein (Fig 3; Sanchez et al, 1999; Wang et al, 2001), preventing the separation of sister chromatids. Moreover, Rad53 can influence the mitotic spindle assembly checkpoint (SAC) through inhibition of the polo-like kinase Cdc5 (Fig 3; Sanchez et al, 1999; Valerio-Santiago et al, 2013; Zhang et al, 2009). Rad53-dependent inactivation of Cdc5, particularly in response to telomere damage, prevents Cdc5's phosphorylation of the spindle assembly checkpoint protein Bfa1 (Valerio-Santiago et al, 2013); however, it is unclear whether Rad53 phosphorylates Cdc5 directly (Fig 3). The inhibition of Cdc5 by DNA damage signaling also indirectly influences the resolution of joint molecules prior to M phase, as Cdc5 activity is important for promoting the activity of the Mus81 resolvase (Szakal & Branzei, 2013). Cdc5 has also been implicated in the down-regulation of the DNA damage response (Donnianni et al, 2010; Vidanes et al, 2010). A more in-depth review exploring Cdc5's complex relationship with the DNA damage response in yeast can be found in Botchkarev and Haber (2018). Similar to its budding yeast counterpart, the mammalian polo-like kinase PLK1 is also inhibited by DNA damage signaling, albeit in an indirect manner (Fig 3; Bruinsma et al, 2017; Qin et al, 2013). A critical mediator of cell cycle arrest in humans is the p53 transcription factor, whose classical function is to trigger the apoptotic program (reviewed in Chen, 2016). p53 is directly phosphorylated and stabilized by all the DNA damage signaling kinases, eliciting a p53-dependent transcriptional response that impacts the DNA damage response (reviewed in Kruse & Gu, 2009). p53-mediated expression of the CDK inhibitor protein p21 represents the primary mechanism by which p53 blocks progression through the cell cycle (Fig 3; Harper et al, 1993, 1995). Apical kinases in mammalian DNA damage signaling also phosphorylate the p53 inhibitor Mdm2, impairing its ability to promote p53 nuclear export and its subsequent degradation (Mayo et al, 1997; Maya et al, 2001; Shinozaki et al, 2003). Unlike most factors covered in Fig 3, Saccharomyces cerevisiae does not have clear p53 or Mdm2 homologs. That said, for reasons discussed in the section on dNTP regulation, the DNA damage signaling-mediated control of p53 might functionally resemble the control of the Crt1 transcription regulator by Rad53 and Dun1 (Huang et al, 1998). Fork stability and protection During DNA replication, stalled replication forks may be targeted and degraded by nucleases. Since fork degradation pathways impair replication fork restart after stalling, leading to persistent DNA lesions, they are considered a major driving force of genomic instability (recently reviewed in Pasero & Vindigni, 2017; Patel & Weiss, 2018). In S. cerevisiae, Rad53 is believed to play a major role in fork stability. It is worth mentioning that Rad53 is essential, and that the lethality of rad53 cells can be rescued by deletion of SML1, an inhibitor of the ribonucleotide reductase enzyme responsible for catalyzing the rate-limiting step in DNA precursor synthesis (Zhao et al, 1998). Nonetheless, even in the absence of SML1, Rad53 mutants are exquisitely sensitive to chemical agents that damage DNA or stall DNA replication forks (Allen et al, 1994; Sanchez et al, 1999; Gunjan & Verreault, 2003). This sensitivity to DNA damaging agents has been primarily attributed to Rad53's role in stabilizing and restarting stalled replication forks (reviewed in detail here: Segurado & Tercero, 2009). Rad53 protects stalled replication forks from nucleolytic processing by phosphorylating and inhibiting the Exo1 exonuclease (Fig 3; Cotta-Ramusino et al, 2005; Morin et al, 2008; Segurado & Diffley, 2008). In addition, Rad53 regulates the Pif1 and Rrm3 helicases, potentially limiting replication fork reversal and subsequent fork degradation (Fig 3; Rossi et al, 2015). However, precisely how DNA damage signaling maintains replication fork integrity is unknown and represents a fundamental knowledge gap in the field. Recent work has revealed that Rad53 phosphorylates the replicative helicase component Cdc45, which in turn recruits and stabilizes Rad53 at replication complexes (Can et al, 2018). Identification of additional key substrates and recruiting mechanisms will be necessary to deconstruct Rad53's functions in maintaining fork stability, which may involve a range of redundant phosphorylation events in more than one essential replisome protein. Mammalian DNA damage signaling kinases also play key roles in protecting stalled replication forks (Fig 3). Human EXO1 is phosphorylated by ATR, which promotes ubiquitylation-dependent degradation of EXO1 and prevents chromosome fragmentation due to unrestrained EXO1 processing activity (El-Shemerly et al, 2008; Tomimatsu et al, 2017). CHK1 also phosphorylates EXO1 directly on serine 746, creating a docking site for binding to 14-3-3 proteins, which prevent recruitment of EXO1 to chromatin and limit EXO1 action at stalled forks (Engels et al, 2011; Li et al, 2019). In addition, ATR governs the recruitment and/or stability of several helicases important for remodeling the stalled fork and promoting fork restart. For example, ATR phosphorylates the Werner syndrome helicase WRN at multiple S/T-Q sites, promoting WRN-RPA co-localization at replication stress sites (Ammazzalorso et al, 20" @default.
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• W2967538289 title "<scp>DNA</scp>damage kinase signaling: checkpoint and repair at 30 years" @default.
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