Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae

KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N-terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109Δ mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double-strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.     

DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein–protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint.     

Taming the tiger by the tail: modulation of DNA damage responses by telomeres

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell‐cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell‐cycle division are discussed.     

Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis

DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.     

DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.     

Similarities and differences between “uncapped” telomeres and DNA double-strand breaks

Telomeric DNA is present at the ends of eukaryotic chromosomes and is bound by telomere “capping” proteins, which are the (Cdc13–Stn1–Ten1) CST complex, Ku (Yku70–Yku80), and Rap1–Rif1–Rif2 in budding yeast. Inactivation of any of these complexes causes telomere “uncapping,” stimulating a DNA damage response (DDR) that frequently involves resection of telomeric DNA and stimulates cell cycle arrest. This is presumed to occur because telomeres resemble one half of a DNA double-strand break (DSB). In this review, we outline the DDR that occurs at DSBs and compare it to the DDR occurring at uncapped telomeres, in both budding yeast and metazoans. We give particular attention to the resection of DSBs in budding yeast by Mre11–Xrs2–Rad50 (MRX), Sgs1/Dna2, and Exo1 and compare their roles at DSBs and uncapped telomeres. We also discuss how resection uncapped telomeres in budding yeast is promoted by the by 9–1–1 complex (Rad17–Mec3–Ddc1), to illustrate how analysis of uncapped telomeres can serve as a model for the DDR elsewhere in the genome. Finally, we discuss the role of the helicase Pif1 and its requirement for resection of uncapped telomeres, but not DSBs. Pif1 has roles in DNA replication and mammalian and plant CST complexes have been identified and have roles in global genome replication. Based on these observations, we suggest that while the DDR at uncapped telomeres is partially due to their resemblance to a DSB, it may also be partially due to defective DNA replication. Specifically, we propose that the budding yeast CST complex has dual roles to inhibit a DSB-like DDR initiated by Exo1 and a replication-associated DDR initiated by Pif1. If true, this would suggest that the mammalian CST complex inhibits a Pif1-dependent DDR.     

α‐Synuclein toxicity in yeast and human cells is caused by cell cycle re‐entry and autophagy degradation of ribonucleotide reductase 1

α‐Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2‐dependent growth signaling, cell cycle re‐entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re‐entry and S‐phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age‐related neurodegenerative diseases.     

Influence of ascorbic acid on the activity of the investigational anticancer drug KP1019

Ascorbic acid has been previously discussed to have antitumor potential through its interaction with transition metal ions such as iron and copper. Furthermore, ascorbic acid may act as a reducing agent for Ru(III) compounds such as indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), an investigational anticancer drug which is supposed to be activated by reduction, prior to binding to cellular target proteins. Therefore, we investigated the influence of ascorbic acid on the activity of this antitumor metal complex in cell culture studies. We show that co-incubation of equicytotoxic, constant amounts of KP1019 with high concentrations of ascorbic acid (50–700 μM) increases cytotoxicity of the ruthenium anticancer drug in the human colon carcinoma cell line SW480, human cervical carcinoma KB-3-1 cells, and the multidrug-resistant subline KBC-1, whereas addition of low concentrations (2.7–50 μM) has a strong chemoprotective effect in the human colon carcinoma cell line SW480, but not in multidrug-resistant KBC-1 cells. Although cellular uptake of KP1019 is not altered, ascorbic acid induce stronger interaction of the ruthenium compound with DNA both in SW480 cells and under cell-free conditions with plasmid DNA. Even if DNA interactions probably play a subordinate role in vivo given the extensive protein binding of the compound, our data exemplify that ascorbic acid enhances the reactivity of KP1019 with biomolecules. Moreover, we demonstrate that the levels of KP1019-generated reactive oxygen species are markedly decreased by co-incubation with ascorbic acid. Conclusively, our results indicate that application of high doses of ascorbic acid might increase the anticancer effects of KP1019.     

Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

Stable Cellular Senescence Is Associated with Persistent DDR Activation

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.     

The roles of p27Kip1 and DNA damage signalling in the chemotherapy‐induced delayed cell cycle checkpoint

DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR‐dependent checkpoint response, followed by a later DDR‐independent, but p27^Kip1‐dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA‐PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two‐step response was DDR‐dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA‐PKcs resulted in cell‐cycle re‐entry despite the increased levels of p27^Kip1 at all time points analyzed. We further investigated the regulation of p27^Kip1 protein levels in the particular setting. Our results showed that the protein status of p27^Kip1 is mainly determined by p38‐MAPK, whereas the role of SKP2 is less significant in the doxoroubicin‐treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy‐induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.     

Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events.Key words: DNA damage checkpoint, Ras signaling, budding yeast, cAMP-dependent protein kinase, anaphase promoting complex, neurofibromatosis type 1     

The Mec1p and Tel1p checkpoint kinases allow humanized yeast to tolerate chronic telomere dysfunctions by suppressing telomere fusions

In this work we report that budding yeasts carrying human-type telomeric repeats at their chromosome termini show a chronic activation of the Rad53-dependent DNA damage checkpoint pathway and a G2/M cell cycle delay. Furthermore, in the absence of either TEL1/ATM or MEC1/ATR genes, which encodes phosphatidylinositol 3-kinase-related kinases (PIKKs), we detected telomere fusions, whose appearance correlates with a reduced cell viability and a high rate of genome instability. Based on sequence analysis, telomere fusions occurred primarily between ultrashort telomeres. Microcolony formation assays argue against the possibility that fusion-containing cells are eliminated by PIKK-dependent signalling.These findings reveal that humanized telomeres in yeast cells are sensed as a chronically damaged DNA but do not greatly impair cell viability as long as the cells have a functional DNA damage checkpoint.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x_L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.     

Taking the time to make important decisions: The checkpoint effector kinases Chk1 and Chk2 and the DNA damage response

The cellular DNA damage response (DDR) is activated by many types of DNA lesions. Upon recognition of DNA damage by sensor proteins, an intricate signal transduction network is activated to coordinate diverse cellular outcomes that promote genome integrity. Key components of the DDR in mammalian cells are the checkpoint effector kinases Chk1 and Chk2 (referred to henceforth as the effector kinases; orthologous to spChk1 and spCds1 in the fission yeast S. pombe and scChk1 and scRad53 in the budding yeast S. cerevisiae). These evolutionarily conserved and structurally divergent kinases phosphorylate numerous substrates to regulate the DDR. This review will focus on recent advances in our understanding of the structure, regulation, and functions of the effector kinases in the DDR, as well as their potential roles in human disease.