**6. Bypass of single-strand gaps and replication blocks by template switch mechanisms**

Template switch mechanisms also allow polymerases to bypass replication forks and resume DNA synthesis; these mechanisms are generally thought to occur on both leading and lagging strands. Template switching is orchestrated by proteins that modify the DNA polymerase processivity factor, PCNA. When the high fidelity polymerase stalls at the replication block, Rad18/Rad6 monoubiquitinates PCNA at the K164 position; monoubiquitinated PCNA can facilitate polymerase switching from to a translesion polymerase of lower fidelity and processivity. PCNA may further become polyubiquitinated at position K164 by combined action of Ubc13/ Mms2/Rad5 (for review, see [115]). Rad5 also contains a helicase function that catalyzes replication fork reversal and is required for template switch mechanisms on the lagging strand [116]. While Rad5 does not require DNA damage at the stalled replication fork for recruitment [112], Rad5 over-expression can trigger genome instability [117]. The checkpoint signaling cascade, mediated by the Dun1 kinase, regulates Rad5 at the post-transcriptional level by destabilizing Rad5 mRNA [118]. These studies indicate that *RAD5* function is regulated.

However, checkpoint signaling may also facilitate template switch mechanisms. Rad53 is required for DNA damage-associated unequal SCE after exposure to MMS [119] and Rad53-mediated Rad55 phosphorylation confers enhanced MMS resistance when RAD5 is also defective [25]. The Rad9 checkpoint protein binds to persistent single strand gaps on the lagging strand, inhibiting the RecQ-like Sgs1 anti-recombination function. In addition Pif1, which is phosphorylated by Rad53, functions in template switching [120]. With longer term checkpoint-mediated G2 arrest, however, Rev1 protein levels accumulate [121, 122], suggesting that error-prone polymerases may serve as the ultimate backup in postreplication repair after error-free mechanisms have failed.

## **7. Choice of DNA damage tolerance pathway is influenced by the DNA lesion**

Multiple tolerance pathways can confer resistance to particular types of DNA damage and the pathway preference depends on the DNA damaging agent. For example, MMS exposure generates by 7 Me-Guanine and 3 Me-Adenine lesions; while the <sup>7</sup> Me-Guanine is mutagenic, the <sup>3</sup> Me-Adenine blocks replication [123]. Replication bypass can occur by error-prone or error-free polymerases, or by template switching. While all three pathways are involved in bypass of 3 Me-Adenine lesions [124], template switch mechanisms are preferred [125]. Checkpoint signaling facilitates template switch mechanisms after exposure to MMS [125, 126]. These studies suggest that template switch mechanisms may be the preferred pathway for bypassing particular lesions that block DNA replication.

The preference of template switch mechanisms or translesion pathways may depend on the efficiency of bypass and repair for large bulky adduct or cross-links. Particular UV-associated DNA cross-links are efficiently bypassed using either polε [127] or a two-step mechanism involving polε and polζ [128]. However, error-free bypass of 4–6 pyrmidine-pyrimidone lesions, present on a plasmid, occurs by template switch mechanisms after their introduction in a NER deficient yeast strain [129]. Likewise, 4-NQO induces bulky damage and stimulates template switch mechanisms [126]. These studies indicate that template switch mechanisms are likely used in error-free postreplication repair pathways [130].

#### **8. Attenuation of the S phase checkpoint activation**

In order for the cell cycle to resume and chromatids to separate the checkpoint activation needs to be downregulated and joint molecules need to be resolved. Once replication is completed, Mrc1 functions as an adaptor for Mec1-mediated checkpoint signaling is diminished since there are no more replication forks [33].

**81**

**Figure 3.**

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

Rad53 hyperphosphorylation.

not hinder sister chromatid division [134].

Rad53 dominant lethality mutant [69].

Resumption of the cell cycle is accomplished by dephosphorylating Rad53 [131]. However, single-strand gaps on sister chromatids can still function to trigger Rad9 mediated checkpoint signaling. To dampen Rad9's adaptor function in mediating Mec1 catalyzed Rad53 phosphorylation, competitive scaffolds compete with Rad9 binding to chromatin [132]. For example, the Mec1-mediated phosphorylation of Slx4 enables an association with Rtt107/Dpb11, which provides a competitive scaffold for the interaction of Rad9 with Dpb11 [133]. These mechanisms thus prevent

Cleavage of joint DNA strands, or Holliday structures, is timed just before anaphase so that cleavage does not occur during S phase. Both kinases and phosphatases fine tune the timing of joint molecule cleavage. Cdk phosphorylates structurespecific nucleases Slx1/Slx4 and Mus81/Mus4 in late G2 and M phases respectively [134]. Whereas Mec1 phosphorylates and subsequently inactivates Yen1, Cdc14 dephosphorylates the inactivated form in mitosis, ensuring that joint molecules do

Similar to adaptions to DSBs, phosphatases deactivate Rad53 (**Figure 3**). These phosphatases include Pph3/Psy2 complex and Ptc1, 2. Interestingly, Pph3 directly interacts with Mec1/Ddc2 [135] at the replication fork, although the interaction does not rely on DNA damage [135]. Besides Rad53, other Mec1 substrates are likely dephosphorylated by Pph3, including phosphorylated Mec1. Thus Pph3 could potentially upregulate Mec1. However, the full range of Pph3 substrates is unknown [135].

Mutations in different phosphatases may confer sensitivities to different DNA damaging agents (**Table 2**). For example, *pph3* and *psy2* mutants are hypersensitive to phelomycin but not 4NQO, while *ptc2*, *ptc3* and *ptc2 ptc3* double mutants are not phleomycin sensitive and are not required for recovery from MMS-associated checkpoint delay [136, 142]. On the other hand *ptc2 ptc3* double mutants are hypersensitive to 4-NQO while *pph3* and *psy2* mutants are not sensitive. However, for particular agents, such as cisplatin, the triple *pph3 ptc2 ptc3* mutant, is synergistically more sensitive [140]. One idea is that phosphorylation of Rad53 is differentially patterned by particular DNA damaging agents, and that the phosphatases,

Ptc2/Ptc3 and Pph3/Psy2 recognize different patterns [142]. This notion is supported by the identification of different MMS and 4-NQO associated Rad53 phosphorylation sites. The connection between Ptc2 and checkpoint activation is further strengthened by observations that over-expression of Ptc2 suppresses the lethality in a

*Double-strand breaks, collapsed replication forks, and replication stress lead to checkpoint activation. Activated Rad53 is dephosphorylated by a series of phosphatases, depending on the signal induced by the DNA damaging agent, shown below the DNA damage. The 5′ to 3′ polarity of the DNA is designated by an arrow. The* 

*substrates of the phosphatases Pph3/Psy2 and Ptc2/Ptc3 include Mec1, Rad53, and Exo1.*

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

*Saccharomyces*

polymerase stalls at the replication block, Rad18/Rad6 monoubiquitinates PCNA at the K164 position; monoubiquitinated PCNA can facilitate polymerase switching from to a translesion polymerase of lower fidelity and processivity. PCNA may further become polyubiquitinated at position K164 by combined action of Ubc13/ Mms2/Rad5 (for review, see [115]). Rad5 also contains a helicase function that catalyzes replication fork reversal and is required for template switch mechanisms on the lagging strand [116]. While Rad5 does not require DNA damage at the stalled replication fork for recruitment [112], Rad5 over-expression can trigger genome instability [117]. The checkpoint signaling cascade, mediated by the Dun1 kinase, regulates Rad5 at the post-transcriptional level by destabilizing Rad5 mRNA [118].

However, checkpoint signaling may also facilitate template switch mechanisms.

error-prone polymerases may serve as the ultimate backup in postreplication repair

Multiple tolerance pathways can confer resistance to particular types of DNA damage and the pathway preference depends on the DNA damaging agent. For

Me-Guanine and 3

Me-Adenine lesions;

Me-Adenine

Me-Adenine blocks replication [123].

**7. Choice of DNA damage tolerance pathway is influenced by the** 

Replication bypass can occur by error-prone or error-free polymerases, or by template switching. While all three pathways are involved in bypass of 3

lesions [124], template switch mechanisms are preferred [125]. Checkpoint signaling facilitates template switch mechanisms after exposure to MMS [125, 126]. These studies suggest that template switch mechanisms may be the preferred pathway for

The preference of template switch mechanisms or translesion pathways may depend on the efficiency of bypass and repair for large bulky adduct or cross-links. Particular UV-associated DNA cross-links are efficiently bypassed using either polε [127] or a two-step mechanism involving polε and polζ [128]. However, error-free bypass of 4–6 pyrmidine-pyrimidone lesions, present on a plasmid, occurs by template switch mechanisms after their introduction in a NER deficient yeast strain [129]. Likewise, 4-NQO induces bulky damage and stimulates template switch mechanisms [126]. These studies indicate that template switch mechanisms are

In order for the cell cycle to resume and chromatids to separate the checkpoint activation needs to be downregulated and joint molecules need to be resolved. Once replication is completed, Mrc1 functions as an adaptor for Mec1-mediated checkpoint signaling is diminished since there are no more replication forks [33].

Rad53 is required for DNA damage-associated unequal SCE after exposure to MMS [119] and Rad53-mediated Rad55 phosphorylation confers enhanced MMS resistance when RAD5 is also defective [25]. The Rad9 checkpoint protein binds to persistent single strand gaps on the lagging strand, inhibiting the RecQ-like Sgs1 anti-recombination function. In addition Pif1, which is phosphorylated by Rad53, functions in template switching [120]. With longer term checkpoint-mediated G2 arrest, however, Rev1 protein levels accumulate [121, 122], suggesting that

These studies indicate that *RAD5* function is regulated.

after error-free mechanisms have failed.

example, MMS exposure generates by 7

Me-Guanine is mutagenic, the <sup>3</sup>

bypassing particular lesions that block DNA replication.

likely used in error-free postreplication repair pathways [130].

**8. Attenuation of the S phase checkpoint activation**

**DNA lesion**

while the <sup>7</sup>

**80**

Resumption of the cell cycle is accomplished by dephosphorylating Rad53 [131]. However, single-strand gaps on sister chromatids can still function to trigger Rad9 mediated checkpoint signaling. To dampen Rad9's adaptor function in mediating Mec1 catalyzed Rad53 phosphorylation, competitive scaffolds compete with Rad9 binding to chromatin [132]. For example, the Mec1-mediated phosphorylation of Slx4 enables an association with Rtt107/Dpb11, which provides a competitive scaffold for the interaction of Rad9 with Dpb11 [133]. These mechanisms thus prevent Rad53 hyperphosphorylation.

Cleavage of joint DNA strands, or Holliday structures, is timed just before anaphase so that cleavage does not occur during S phase. Both kinases and phosphatases fine tune the timing of joint molecule cleavage. Cdk phosphorylates structurespecific nucleases Slx1/Slx4 and Mus81/Mus4 in late G2 and M phases respectively [134]. Whereas Mec1 phosphorylates and subsequently inactivates Yen1, Cdc14 dephosphorylates the inactivated form in mitosis, ensuring that joint molecules do not hinder sister chromatid division [134].

Similar to adaptions to DSBs, phosphatases deactivate Rad53 (**Figure 3**). These phosphatases include Pph3/Psy2 complex and Ptc1, 2. Interestingly, Pph3 directly interacts with Mec1/Ddc2 [135] at the replication fork, although the interaction does not rely on DNA damage [135]. Besides Rad53, other Mec1 substrates are likely dephosphorylated by Pph3, including phosphorylated Mec1. Thus Pph3 could potentially upregulate Mec1. However, the full range of Pph3 substrates is unknown [135].

Mutations in different phosphatases may confer sensitivities to different DNA damaging agents (**Table 2**). For example, *pph3* and *psy2* mutants are hypersensitive to phelomycin but not 4NQO, while *ptc2*, *ptc3* and *ptc2 ptc3* double mutants are not phleomycin sensitive and are not required for recovery from MMS-associated checkpoint delay [136, 142]. On the other hand *ptc2 ptc3* double mutants are hypersensitive to 4-NQO while *pph3* and *psy2* mutants are not sensitive. However, for particular agents, such as cisplatin, the triple *pph3 ptc2 ptc3* mutant, is synergistically more sensitive [140]. One idea is that phosphorylation of Rad53 is differentially patterned by particular DNA damaging agents, and that the phosphatases, Ptc2/Ptc3 and Pph3/Psy2 recognize different patterns [142]. This notion is supported by the identification of different MMS and 4-NQO associated Rad53 phosphorylation sites. The connection between Ptc2 and checkpoint activation is further strengthened by observations that over-expression of Ptc2 suppresses the lethality in a Rad53 dominant lethality mutant [69].

#### **Figure 3.**

*Double-strand breaks, collapsed replication forks, and replication stress lead to checkpoint activation. Activated Rad53 is dephosphorylated by a series of phosphatases, depending on the signal induced by the DNA damaging agent, shown below the DNA damage. The 5′ to 3′ polarity of the DNA is designated by an arrow. The substrates of the phosphatases Pph3/Psy2 and Ptc2/Ptc3 include Mec1, Rad53, and Exo1.*


#### **Table 2.**

*Phosphatases that function in checkpoint adaptation to specific DNA damaging agents.*

Tolerance to MMS-induced DNA damage includes reactivation of stalled replication forks, which depends on the level of Rad53 phosphorylation [143]. Pph3/Psy2 phosphatase is the principle phosphatase that deactivates Rad53. In the absence Pph3/Psy2 replication restart can occur; however late origins are used to complete DNA replication. Interestingly, downregulation of Rad53 phosphorylation by a HA-Rad53 or a *dot1* deletion confers higher levels of MMS resistance, although at the sake of more Rev1 foci and mutagenesis [144]. These studies would suggest that MMS-induced checkpoint activation is a double-edged sword; limiting MMS-induced mutation may come at the cost of toxic recombination intermediates.

While tolerance to MMS-induced DNA damage relies on dampening the checkpoint response, UV resistance heavily relies on checkpoint activation, as illustrated by observations that the *rad14 mec1* double mutant, defective in both NER and checkpoint signaling, is synergistically more UV sensitive [145]. In yeast, UV triggers the G1-S checkpoint when NER is functional, but unrepaired UV lesions trigger checkpoint responses in S and G2 cells [146]. Interestingly, chronic exposure to low dose UV does not elicit cell cycle arrest at the G2 checkpoint, suggesting that DNA replication machinery is not significantly impeded during chronic exposure [147].

**83**

tion and for adaptation.

4NQO [137].

**checkpoint response** 

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

exhibit higher frequencies of AFB1-associated mutations.

**9. Nutrient sensing and the regulation of adaptation and the** 

diploid strains exposed to DNA damaging agents [156].

One unifying theme in DNA damage tolerance to multiple types of DNA lesions is that nutrient sensing plays an important role in promoting downregulation of the checkpoint response. Deregulation of *IRA1* and *IRA2*, which control glucosegrowth signaling, prevent adaptation to uncapped telomeres in *cdc13* strains [155]. Inhibition of TOR1 by rapamycin prevents adaptation and aneuploidy in rad52

Nutrient sensing is also important in controlling the checkpoint response through type 2A protein phosphatases. In the presence of plentiful carbon and nitrogen, target of rapamycin (TORC1) activates Mec1-signaling pathway by inhibiting PP2A and PP2A-like phosphatases. PP2A activators include ceramide and S-adenosyl methionine (SAM) [139]. The effect of this signaling on the PP2C and PP4 phosphatases is unclear. Nonetheless, these studies illustrate that the DNA damage response requires an active growth signaling response [139]. Recent data also suggests that TORC1 inhibition results in lower levels of checkpoint proteins [157]. Thus, it may appear that TORC1 may be required for both checkpoint activa-

P450-acitvated carcinogens may also elicit a strong DNA damage inducible effect. For example, aflatoxin B1 (AFB1), induces strong Rad53 activation in budding yeast, which generally occurs within two hours of exposure and then is gradually attenuated [148]). AFB1 exposure also upregulates the expression of DNA repair genes, including Rad51, Csm2, and Rad16 [149, 150]. Interestingly, AFB1 exposure elicits an S phase delay coinciding with the appearance of Rad51 foci [148]. This is consistent with AFB1 being a strong recombinagen but weak mutagen in yeast [151]. Interestingly, checkpoint signaling is required for stimulation of both AFB1-associated unequal sister chromatid recombination and mutation [152]. By profiling the yeast genome for AFB1 resistance using next generation sequencing, St. John *et al.* [138] identified both HR genes, including Rad54, Rad55, and Csm2, and those encoding error-prone polymerases. Similar to alkylated induced damage, the Csm2(Shu) complex favors an error-free template switch mechanism [153]; thus, csm2 mutants are deficient in sister chromatid recombination but

Genes the confer AFB1 resistance included *PSY3*, *CKB1* and *CKB2*, which function in DNA damage tolerance [138]. While the genes encoding the CKII substrates, Ptc2 and Ptc3, did not appear in the screen, the identification of both CKII and Pph3 suggest that tolerance to AFB1-associated DNA damage requires both phosphorylation and dephosphorylation of multiple proteins. The identity of these proteins may further elucidate how AFB1-associated DNA damage is tolerated. Additional phosphatases that function in DNA damage tolerance include PP2A and PP2A-like phosphatases. These phosphatases are composed of catalytic subunits, such as Pph21 and Pph22, scaffolding subunits, and regulatory subunits, such as Cdc55 and Rts1. While a direct interaction with phosphorylated Rad53 has not been demonstrated, the PP2A phosphatase suppresses the checkpoint response after HU exposure [139]. While the identity of all of the PP2A substrates is unknown, PP2A is involved in both cytokinesis and mitosis [154]. Particular regulatory subunits are required for tolerance to different DNA damaging agents. For example, Rts1 is required for DNA damage tolerance after *rad51* cells are exposed to bleomycin [141] and Sit4, a PP2A-like phosphatase, is required for tolerance to

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

*Saccharomyces*

Restrictive temperature for *cdc13*

4-Nitroquinoline

mutant

oxide

Methyl methanesulfonate

**DNA damaging agent or environmental condition**

**82**

intermediates.

**Table 2.**

chronic exposure [147].

Tolerance to MMS-induced DNA damage includes reactivation of stalled replication forks, which depends on the level of Rad53 phosphorylation [143]. Pph3/Psy2 phosphatase is the principle phosphatase that deactivates Rad53. In the absence Pph3/Psy2 replication restart can occur; however late origins are used to complete DNA replication. Interestingly, downregulation of Rad53 phosphorylation by a HA-Rad53 or a *dot1* deletion confers higher levels of MMS resistance, although at the sake of more Rev1 foci and mutagenesis [144]. These studies would suggest that MMS-induced checkpoint activation is a double-edged sword; limiting MMS-induced mutation may come at the cost of toxic recombination

**DNA damage Phosphatase**

HO Endonuclease Double-strand break Ptc2/Ptc3 Completely

Bulky adduct and oxidative

Major and minor groove

formamidopyrimidine

Pyrimidine-pyrimidone

forks, double-strand breaks

*Phosphatases that function in checkpoint adaptation to specific DNA damaging agents.*

Camptothecin Topo1 cross-link with DNA Ptc2, Ptc3 Synergistically

Cisplatin DNA cross links Pph3/Psy2 Synergistically

damage

strand breaks

alkylations

cross links

Phleomycin Single-strand and double-

Aflatoxin B1 AFB1-N7-Gua, and AFB1

Ultraviolet radiation Pyrimidine dimers and

Hydroxyurea Stalled DNA replication

Bleomycin Single and double-strand break s

**Required for Resistance or Adaptation**

Long tracts of ssDNA Ptc2 Unknown [84]

Pph3, Ptc2/Ptc3

Pph3/Psy2, Pph2

Pph3, Rts1 (regulator of Cdc55)

**Phenotype of Δ** *ptc2 ptc3 pph3*

deficient in adaptation

Ptc2/Ptc3, Sit4 Unknown [136, 137]

sensitive

Pph3/Psy2 Unknown [138]

sensitivity

sensitive

sensitive

Synergistically sensitive

Pph3/Psy2 Synergistically

Not required Moderate

Unknown [136]

**Reference**

[70]

[70]

[70]

[70]

[140]

Unknown [73, 141]

[70, 139]

While tolerance to MMS-induced DNA damage relies on dampening the checkpoint response, UV resistance heavily relies on checkpoint activation, as illustrated by observations that the *rad14 mec1* double mutant, defective in both NER and checkpoint signaling, is synergistically more UV sensitive [145]. In yeast, UV triggers the G1-S checkpoint when NER is functional, but unrepaired UV lesions trigger checkpoint responses in S and G2 cells [146]. Interestingly, chronic exposure to low dose UV does not elicit cell cycle arrest at the G2 checkpoint, suggesting that DNA replication machinery is not significantly impeded during

P450-acitvated carcinogens may also elicit a strong DNA damage inducible effect. For example, aflatoxin B1 (AFB1), induces strong Rad53 activation in budding yeast, which generally occurs within two hours of exposure and then is gradually attenuated [148]). AFB1 exposure also upregulates the expression of DNA repair genes, including Rad51, Csm2, and Rad16 [149, 150]. Interestingly, AFB1 exposure elicits an S phase delay coinciding with the appearance of Rad51 foci [148]. This is consistent with AFB1 being a strong recombinagen but weak mutagen in yeast [151]. Interestingly, checkpoint signaling is required for stimulation of both AFB1-associated unequal sister chromatid recombination and mutation [152]. By profiling the yeast genome for AFB1 resistance using next generation sequencing, St. John *et al.* [138] identified both HR genes, including Rad54, Rad55, and Csm2, and those encoding error-prone polymerases. Similar to alkylated induced damage, the Csm2(Shu) complex favors an error-free template switch mechanism [153]; thus, csm2 mutants are deficient in sister chromatid recombination but exhibit higher frequencies of AFB1-associated mutations.

Genes the confer AFB1 resistance included *PSY3*, *CKB1* and *CKB2*, which function in DNA damage tolerance [138]. While the genes encoding the CKII substrates, Ptc2 and Ptc3, did not appear in the screen, the identification of both CKII and Pph3 suggest that tolerance to AFB1-associated DNA damage requires both phosphorylation and dephosphorylation of multiple proteins. The identity of these proteins may further elucidate how AFB1-associated DNA damage is tolerated.

Additional phosphatases that function in DNA damage tolerance include PP2A and PP2A-like phosphatases. These phosphatases are composed of catalytic subunits, such as Pph21 and Pph22, scaffolding subunits, and regulatory subunits, such as Cdc55 and Rts1. While a direct interaction with phosphorylated Rad53 has not been demonstrated, the PP2A phosphatase suppresses the checkpoint response after HU exposure [139]. While the identity of all of the PP2A substrates is unknown, PP2A is involved in both cytokinesis and mitosis [154]. Particular regulatory subunits are required for tolerance to different DNA damaging agents. For example, Rts1 is required for DNA damage tolerance after *rad51* cells are exposed to bleomycin [141] and Sit4, a PP2A-like phosphatase, is required for tolerance to 4NQO [137].
