**2. Checkpoint activation initiated by DSBs**

Checkpoint activation triggered by DSBs, and subsequent recovery or adaptation has been extensively studied in strains containing either uncapped telomeres or chromosomal DSBs that cannot be repaired by HR. An unrepaired DSB occurs when HO endonuclease cleaves the recognition sequence at the MAT locus but silent mating type locus has been deleted [34]. Uncapped telomeres occur when either the Cdc13-Stn1-Ten1 (CST) complex or the Ku complex, composed of yKu70 and yKu80, is defective. At restrictive (elevated) temperatures in either *cdc13* or *yku70* mutants, extensive tracts of single-stranded DNA complex are generated [35]. While two DNA ends are revealed by a single unrepaired DSB at the MAT locus, at the non-permissive temperature in *cdc13* mutants single-stranded DNA is revealed at the telomeres of sixteen chromosomes, thus amplifying the DNA damage signal.

A single DSB occurring in G1 does not trigger cell cycle arrest at the G1/S or intra S phase checkpoint [36], but instead the cell cycle progresses through S phase and into G2 phase, where cells arrest. Repair of DSBs can occur at any time in the cell cycle by NHEJ; however, in budding yeast, NHEJ is favorable when the singlestrand overhangs are short [37]. However, DSBs, will trigger a partial DNA damage response in G1 cells [38], and recombination proteins, such as Rad51 and Rad54, are still induced [39] and Rad55 is phosphorylated [38] .

The orchestration of checkpoint signaling has been well described in current reviews [40] and is briefly summarized (**Figure 1**). Mre11/Rad50/Xrs2 (MRX) and Tel1 (ataxia telangiectasia mutated (ATM) ortholog) bind to the ends of the DSB, which facilitates the juxtaposition of the ends of the breaks. NHEJ requires yKu70 and yKu80. However, if NHEJ is not successful, cyclin dependent kinase I (Cdk1 or Cdc28), which has high activity in G2, phosphorylates Sae2 and the 5′ to 3′ exonuclease Dna2 [41]. Sae2 phosphorylation activates the Mre11 endonuclease activity that ejects yKu70 from the ends of the DSB. Together with Sgs1/Dna2 and Exo1, the ends are further degraded in a 5′ to 3′ direction. NHEJ and resection require chromatin remodeling factors, including the Ino80 complex [42, 43], Rsc complex [44], and Fun30 [45, 46]. Resection is generally slow and proceeds at 1–2 nucleotide per minute [40]. Resection of the ends reveals single stranded DNA (ssDNA), which is then coated by single strand binding protein (RPA), which serves as a general sensor for DNA damage. The RPA-coated ssDNA is a binding site for Ddc2-Mec1 (ataxia telangiectasia mutated and rad3-related (ATR ortholog). Rad24/Rfc facilitates the binding of trimeric Rad17/Mec3/Ddc1 (9–1-1) protein which recognizes the junction between the single stranded DNA and the double-stranded DNA [47]. Thus, ssDNA serves as a general signal for checkpoint signaling [48].

#### **Figure 1.**

*A pathway for checkpoint pathway commences with a DNA damage signal that triggers the PIKK kinase, Mec1. Downstream checkpoint kinases are activated, as facilitated by the adaptors Rad9 and Mrc1. Kinase substrates are identified for Rad53 and Dun1, but both Mec1 and Rad53 phosphorylate multiple substrates that are not shown.*

Mec1, a sensor or apical serine/threonine kinase phosphorylates downstream kinases, DNA repair proteins, and histones, preferably at SQ/TQ sites [49]. Both Mec1 and Tel1 phosphorylate histone γ-H2A for ~50 kb on either side of the DSB, which serves to recruit other checkpoint protein, such as the adaptor, Rad9 (53BP1 ortholog). Mec1 regulates checkpoint signaling by autophosphorylation on the S1964 residue [50] and phosphorylation of Ddc2, which destabilizes unbound Ddc2 and limits the amount of bound Ddc2-Mec1. Mec1 also phosphorylates Exo1 [51], which limits the amount of single-stranded DNA that could serve as a signal for checkpoint activation. Thus, Mec1's activity serves to not only activate downstream kinases but also dampen the checkpoint response.

Rad9, as an adaptor protein and 53BP1 ortholog, is required to bring the effector (transducer) kinases in contact with Mec1. Rad9 binding to chromatin is mediated by its BRCT and tudor domains that interact with phosphorylated and trimethylated histone H3, respectively [52]. While histone phosphorylation is induced by DNA damage, Dot1-mediated histone H3 methylation is constitutive [53]. Localization to damaged DNA is facilitated by binding to Rtt107/Dbp11. Both Mec1 and Cdk1 phosphorylate Rad9 on separate domains [40]. In turn, oligomers of phosphorylated Rad9 bind to Rad53 and facilitate Mec1-mediated phosphorylation. A Rad53 phosphorylated heterodimer then autophosphorylates; the hyper-phosphorylated Rad53 can, in turn, rapidly diffuse throughout the nucleus and phosphorylate multiple substrates, including Dun1 and Asf1. Similarly, Rad9 facilitates Mec1-mediated Chk1 phosphorylation; the activated Chk1 phosphorylates Pds1, which prevents its degradation by the anaphase promoting complex (APC). In turn, sister chromatid cohesion is maintained and anaphase is prevented [54]. Activated Rad53 also inhibits the APC from degrading securin [54]. A Rad53-mediated pathway inhibits Cdc5, a polo-like kinase that functions in mitotic exit through the regulation of spindle pole body separation [30]. While Pds1 phosphorylation can also be triggered by the Mad2-mediated spindle checkpoints, Rad53 phosphorylation is only triggered by DNA damage or stalled replication forks. While the single mutants are partially defective in DNA damage-induced G2 arrest, *rad53 pds1* and *rad53 dun1* double mutants are fully deficient [55].

Partial to full checkpoint activation will also occur when DNA damage processing is rendered less efficient. For example, mating-type switching in a *rad1* mutant, defective in removal of 3′ non-homologous ends, will trigger a

**75**

*Checkpoint Control of DNA Repair in Yeast DOI: http://dx.doi.org/10.5772/intechopen.96966*

be determined (**Table 1**).

Checkpoint Signaling

**Protein Phosphorylated**

Nucleases

Transcription inhibitors

Recombination Proteins

Protein Inhibitors

checkpoint-dependent cell cycle delay [56]. Interestingly, Rad53 phosphorylation was not abundant in the *rad1* mutant; however, the cell cycle delay was not observed in the *rad9* strain, and was shortened in the *mad3* mutants, defective in spindle checkpoint. The authors speculated that checkpoint activation occurred when H2A phosphorylation extended through centromeric chromatin, triggering a spindle pole checkpoint response [56]. These studies indicate that spindle pole checkpoints also participate in the DNA damage response, depending on the context of the DSB. While the G2 checkpoint is critical for HR repair of DSBs, Mec1 and Mec1 signaling pathway also phosphorylate additional DNA repair functions that facilitate DSB repair and damage incurred by radiomimetic agents [40]. For example, Mec1 phosphorylates Rad51 [57] and Rad55 [58]. Phosphorylation of Rad51 enhances its activity and is required for resistance to recombinagens, such as methyl methane sulfonate (MMS) [57]. In addition, Mec1 phosphorylates Slx4, which binds to Rad1/Rad10 and facilitates single-strand annealing by cleaving non-homologous tails [59]. The studies indicate that the checkpoint pathway directly phosphorylates repair proteins to enhance their function. While there are many proteins that are phosphorylated in response to DNA damage [60, 61], the functional significance of the phosphorylation of many of these proteins has yet to

**Kinase Effect Reference**

subsequent Rnr transcriptional activation

and release from Rnr1 subunit and subsequent

[60, 63]

[27]

Rad9 Mec1 /Tel1 Rad53 docking and Rad9 multimerization [40, 60] Rad53 Mec1 Activation of Rad53 autophosphorylation [40, 60] Rad53 Tel1 Activation of Rad53 [40, 60] Chk1 Mec1 Phosphorylation of Pds1 [30, 54] Pds1 Chk1 APC-associated degradation of Pds1 is inhibited [54, 60] Mec1-Ddc2 Mec1 Attenuation of Mec1 kinase activity [50] Dun1 Rad53 Activation of Dun1 kinase activity [40]

Sae2 Cdk1 Cell cycle regulation limiting resection to G2/M [41] Dna2 Cdk1 Cell cycle regulation limiting resection to G2/M [62] Exo1 Mec1 Inhibition of Exo1 5′-3′ exonuclease activity [51]

increase in dNTPs

Dif1 Dun1 Allows for transport of RNR into the cytoplasm [64]

Rad55 Rad53 Enhances recombination in rad5 mutants [58] Rad51 Mec1/Rad53 Enhances activity [57] Rev1 Mec1 Facilitates binding to ssDNA [26]

Crt1 Dun1 Crt1 phosphorylation leads to degradation, and

Sml1 Dun1 Sml1 phosphorylation leads to degradation

#### *Checkpoint Control of DNA Repair in Yeast DOI: http://dx.doi.org/10.5772/intechopen.96966*

*Saccharomyces*

**Figure 1.**

*not shown.*

Mec1, a sensor or apical serine/threonine kinase phosphorylates downstream kinases, DNA repair proteins, and histones, preferably at SQ/TQ sites [49]. Both Mec1 and Tel1 phosphorylate histone γ-H2A for ~50 kb on either side of the DSB, which serves to recruit other checkpoint protein, such as the adaptor, Rad9 (53BP1 ortholog). Mec1 regulates checkpoint signaling by autophosphorylation on the S1964 residue [50] and phosphorylation of Ddc2, which destabilizes unbound Ddc2 and limits the amount of bound Ddc2-Mec1. Mec1 also phosphorylates Exo1 [51], which limits the amount of single-stranded DNA that could serve as a signal for checkpoint activation. Thus, Mec1's activity serves to not only activate downstream

*A pathway for checkpoint pathway commences with a DNA damage signal that triggers the PIKK kinase, Mec1. Downstream checkpoint kinases are activated, as facilitated by the adaptors Rad9 and Mrc1. Kinase substrates are identified for Rad53 and Dun1, but both Mec1 and Rad53 phosphorylate multiple substrates that are* 

Rad9, as an adaptor protein and 53BP1 ortholog, is required to bring the effec-

tor (transducer) kinases in contact with Mec1. Rad9 binding to chromatin is mediated by its BRCT and tudor domains that interact with phosphorylated and trimethylated histone H3, respectively [52]. While histone phosphorylation is induced by DNA damage, Dot1-mediated histone H3 methylation is constitutive [53]. Localization to damaged DNA is facilitated by binding to Rtt107/Dbp11. Both Mec1 and Cdk1 phosphorylate Rad9 on separate domains [40]. In turn, oligomers of phosphorylated Rad9 bind to Rad53 and facilitate Mec1-mediated phosphorylation. A Rad53 phosphorylated heterodimer then autophosphorylates; the hyper-phosphorylated Rad53 can, in turn, rapidly diffuse throughout the nucleus and phosphorylate multiple substrates, including Dun1 and Asf1. Similarly, Rad9 facilitates Mec1-mediated Chk1 phosphorylation; the activated Chk1 phosphorylates Pds1, which prevents its degradation by the anaphase promoting complex (APC). In turn, sister chromatid cohesion is maintained and anaphase is prevented [54]. Activated Rad53 also inhibits the APC from degrading securin [54]. A Rad53-mediated pathway inhibits Cdc5, a polo-like kinase that functions in mitotic exit through the regulation of spindle pole body separation [30]. While Pds1 phosphorylation can also be triggered by the Mad2-mediated spindle checkpoints, Rad53 phosphorylation is only triggered by DNA damage or stalled replication forks. While the single mutants are partially defective in DNA damage-induced G2 arrest, *rad53 pds1* and

Partial to full checkpoint activation will also occur when DNA damage processing is rendered less efficient. For example, mating-type switching in a *rad1* mutant, defective in removal of 3′ non-homologous ends, will trigger a

kinases but also dampen the checkpoint response.

*rad53 dun1* double mutants are fully deficient [55].

**74**

checkpoint-dependent cell cycle delay [56]. Interestingly, Rad53 phosphorylation was not abundant in the *rad1* mutant; however, the cell cycle delay was not observed in the *rad9* strain, and was shortened in the *mad3* mutants, defective in spindle checkpoint. The authors speculated that checkpoint activation occurred when H2A phosphorylation extended through centromeric chromatin, triggering a spindle pole checkpoint response [56]. These studies indicate that spindle pole checkpoints also participate in the DNA damage response, depending on the context of the DSB.

While the G2 checkpoint is critical for HR repair of DSBs, Mec1 and Mec1 signaling pathway also phosphorylate additional DNA repair functions that facilitate DSB repair and damage incurred by radiomimetic agents [40]. For example, Mec1 phosphorylates Rad51 [57] and Rad55 [58]. Phosphorylation of Rad51 enhances its activity and is required for resistance to recombinagens, such as methyl methane sulfonate (MMS) [57]. In addition, Mec1 phosphorylates Slx4, which binds to Rad1/Rad10 and facilitates single-strand annealing by cleaving non-homologous tails [59]. The studies indicate that the checkpoint pathway directly phosphorylates repair proteins to enhance their function. While there are many proteins that are phosphorylated in response to DNA damage [60, 61], the functional significance of the phosphorylation of many of these proteins has yet to be determined (**Table 1**).



#### **Table 1.**

*Proteins phosphorylated by DDR.*

## **3. Checkpoint recovery and adaptation from double-strand break**

Once cells have repaired the DSB, recovery involves reversal of protein modifications and chromatin restoration. While the DNA damage may no longer be present, protein modifications are still present that signal checkpoint activation. To inactivate the G2/M checkpoint and resume division, Rad53 must be dephosphorylated. Two phosphatases involved in the inactivation of Rad53 include phosphorylated versions of the type 2C protein phosphatases (PP2C), Ptc2 and Ptc3 [68–70]; these phosphatases are also involved in inactivating other stress induced pathways, such as the Hog1-mediated osmotic stress induced pathway [71], while Ptc2 dephosphorylates Cdk1. Casein kinase II (Ck2) phosphorylates Ptc2, which specifically binds to the Rad53 FHA1 domains [72]. Interestingly, CK2 mutants are more defective in adaptation than *ptc2* mutants, suggesting that CK2 may control additional genes involved in adaptation [68].

Pph3, a member of the PP4 family, is important in maintaining full recovery; the triple mutant (*ptc2, ptc3, pph3*) is severely defective in DSB repair when the repair pathway is slow [70]. This may be partially explained by observations that Pph3 functions to dephosphorylate γ-H2A, which serves as a signal for activation of checkpoint proteins, cohesins, and chromatin remodelers [73]. However, the mechanism by with chromatin associated gamma γ-H2A is fully dephosphorylated is still being explored.

Chromatin restoration requires Asf1 and Caf1 which reassemble chromatin on DNA (Kim and Haber [74]). Asf1 binds histone H3 triggering acetylation by the histone acetyltransferase, Rtt109, and further ubiquitylation by Rtt101 [75]. This, in turn promotes the binding of the histone H3 and H4 heterodimer by Caf1. Interestingly, Asf1 also functions to bind Rad53, thus serving a role to sequester dephosphorylated Rad53. Thus Asf1 functions both in reassembling chromatin and stabilizing dephosphorylated Rad53 [75].

If a DSB is not repaired, cells will either resume the cell cycle or die. The resumption of the cell cycle is referred to as adaptation. Similar to recovery, adaptation involves both chromatin remodeling and phosphatases that deactivate the Rad53 kinase and Cdk1 kinase. This adaptation is blocked in *yku80* mutants [69], deficient in NHEJ, and *cdc5-ad*, which is defective in mitotic exit. *Yku80* mutants exhibit twice the rate of resection of the DSBs, resulting in more single-stranded DNA

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*Checkpoint Control of DNA Repair in Yeast DOI: http://dx.doi.org/10.5772/intechopen.96966*

downstream checkpoint effectors [78].

and thus the potential for more Rad53 checkpoint signaling. This interpretation is supported by observations that overexpression of Ptc2 is sufficient to suppress the adaptation defect of both *yku70* and *yku80* [69]. However, the role of resection in checkpoint adaptation is complicated by the identification of chromatin remodelers, such as Fun30 [76–78], which are required for adaptation but enhance resection. One possibility is that Fun30-associated resection in γ -H2A-modified chromatin antagonizes the checkpoint protein Rad9 from binding and signaling

Additional genes function to remove recombination proteins from chromatin. Removal of Rad51 filaments is facilitated by the chromatin modifier Tid1 (Rdh54) [79] and the Srs2 helicase, the former is phosphorylated by Mec1 and the latter is phosphorylated by Cdk1 [67]. Both *rdh54* and *srs2* mutants are defective in adaptation (reference [79, 80]). These studies present additional evidence *MEC1* functions both in the triggering of checkpoint arrest as well as recovery from checkpoint arrest.

While single-stranded DNA is present at telomeres, it is normally "capped" by a RPA-like structure, referred to as Cdc13-Stn1-Ten1 (CST), and by Ku (yKu70/yKu80) complex [35]. During replication, Cdc13 is phosphorylated by Cdk1 and recruits telomerase [81]. Telomere ends are susceptible to nucleases in yeast mutants defective in proteins that bind to chromosome ends, such as *yku70*, and that are defective in recruiting telomerase, such as the *cdc13–1* mutant at the restrictive temperature. Pif1 helicase inhibits telomerase and leads to slow resection at the telomere end [82, 83]. Resection is also slowed by binding of Rif1 and Rap1, which bind specifically to single stranded telomere sequences and inhibit the binding of the checkpoint activators, RPA and Rad24 [84, 85]. In the *cdc13–1* mutant, resection is extensive and largely performed by Exo1, leading to ssDNA bound to RPA, the 9–1-1 complex, and Rad9. Similarly, in *yku70* mutants, ssDNA is generated, but it takes several generations for ssDNA to accumulate [35]. The 9–1-1 complex is apparently not involved in eliciting a checkpoint response but Chk1 activation is required for Exo1-mediated resection [86]. In *yku70* mutants, resected telomeres elicit both a spindle and DNA damage checkpoint activation. However, unlike HO-induced DSBs, Mec1 binding does not lead to rapid resection but rather an inhibition of resection through subsequent binding of Rad9 and Rad53 [87]). Resection of the telomere, in turn, may facilitate recombination or break-induced replication (BIR, [88]) using an undamaged chromosomal end as a template for replication to the end of the chromosome. BIR is facilitated by activated Pif1 [89]. Thus, checkpoint activation at uncapped

**4. Uncapped telomeres, checkpoint activation, and adaptation**

telomeres enables alternative mechanisms of telomere lengthening.

kinases that modulate adaptation.

Adaptation to shortened telomeres was first noted by Sandell and Zakian [90] and require CKII and Cdc5 [91]. CKII directly phosphorylates Ptc2, which is required for tolerating shortened telomeres [92]. In addition, phosphorylated Cdc13 can be dephosphorylated by Pph3/Psy3, resulting in the segregation of uncapped chromosomal ends [35]. Over-expression of Cdc5 also decreases Rad53 phosphorylation [93]. Thus, as in HO-induced DSBs, there are multiple phosphatases and

**5. Intra-S phase checkpoint and stabilization of the replication fork**

The purpose of the intra-S phase checkpoint is to maintain replication fork integrity so that replication can be completed; collapsed replication forks are a

#### *Checkpoint Control of DNA Repair in Yeast DOI: http://dx.doi.org/10.5772/intechopen.96966*

*Saccharomyces*

**Protein Phosphorylated**

Resolvases

Helicases

**Table 1.**

*Proteins phosphorylated by DDR.*

Mus81/Mms4 Cdk1, Dbf4

Cdc7

**3. Checkpoint recovery and adaptation from double-strand break**

Yen1 Cdk1 Inhibits function in S phase by transportation to the cytoplasm

Pif1 Rad53 Inhibits fork unwinding, promotes DNA

Srs2 Cdk1 Promotes adaptation by removal of Rad51

junctions

Rrm3 Rad53 Inhibits fork unwinding [66]

filaments

damage tolerance by HR

Once cells have repaired the DSB, recovery involves reversal of protein modifications and chromatin restoration. While the DNA damage may no longer be present, protein modifications are still present that signal checkpoint activation. To inactivate the G2/M checkpoint and resume division, Rad53 must be dephosphorylated. Two phosphatases involved in the inactivation of Rad53 include phosphorylated versions of the type 2C protein phosphatases (PP2C), Ptc2 and Ptc3 [68–70]; these phosphatases are also involved in inactivating other stress induced pathways, such as the Hog1-mediated osmotic stress induced pathway [71], while Ptc2 dephosphorylates Cdk1. Casein kinase II (Ck2) phosphorylates Ptc2, which specifically binds to the Rad53 FHA1 domains [72]. Interestingly, CK2 mutants are more defective in adaptation than *ptc2* mutants, suggesting that CK2 may control additional genes

**Kinase Effect Reference**

Regulation of cleavage of Holliday and branch

[62]

[66]

[62, 67]

[62, 65]

Pph3, a member of the PP4 family, is important in maintaining full recovery; the triple mutant (*ptc2, ptc3, pph3*) is severely defective in DSB repair when the repair pathway is slow [70]. This may be partially explained by observations that Pph3 functions to dephosphorylate γ-H2A, which serves as a signal for activation of checkpoint proteins, cohesins, and chromatin remodelers [73]. However, the mechanism by with chromatin associated gamma γ-H2A is fully dephosphorylated

Chromatin restoration requires Asf1 and Caf1 which reassemble chromatin on DNA (Kim and Haber [74]). Asf1 binds histone H3 triggering acetylation by the histone acetyltransferase, Rtt109, and further ubiquitylation by Rtt101 [75]. This, in turn promotes the binding of the histone H3 and H4 heterodimer by Caf1. Interestingly, Asf1 also functions to bind Rad53, thus serving a role to sequester dephosphorylated Rad53. Thus Asf1 functions both in reassembling chromatin and

If a DSB is not repaired, cells will either resume the cell cycle or die. The resump-

tion of the cell cycle is referred to as adaptation. Similar to recovery, adaptation involves both chromatin remodeling and phosphatases that deactivate the Rad53 kinase and Cdk1 kinase. This adaptation is blocked in *yku80* mutants [69], deficient in NHEJ, and *cdc5-ad*, which is defective in mitotic exit. *Yku80* mutants exhibit twice the rate of resection of the DSBs, resulting in more single-stranded DNA

**76**

involved in adaptation [68].

is still being explored.

stabilizing dephosphorylated Rad53 [75].

and thus the potential for more Rad53 checkpoint signaling. This interpretation is supported by observations that overexpression of Ptc2 is sufficient to suppress the adaptation defect of both *yku70* and *yku80* [69]. However, the role of resection in checkpoint adaptation is complicated by the identification of chromatin remodelers, such as Fun30 [76–78], which are required for adaptation but enhance resection. One possibility is that Fun30-associated resection in γ -H2A-modified chromatin antagonizes the checkpoint protein Rad9 from binding and signaling downstream checkpoint effectors [78].

Additional genes function to remove recombination proteins from chromatin. Removal of Rad51 filaments is facilitated by the chromatin modifier Tid1 (Rdh54) [79] and the Srs2 helicase, the former is phosphorylated by Mec1 and the latter is phosphorylated by Cdk1 [67]. Both *rdh54* and *srs2* mutants are defective in adaptation (reference [79, 80]). These studies present additional evidence *MEC1* functions both in the triggering of checkpoint arrest as well as recovery from checkpoint arrest.
