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

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 telomeres enables alternative mechanisms of telomere lengthening.

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 kinases that modulate adaptation.

#### **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 major source of genetic instability [94]. Replication forks stall because of limiting amounts of dNTPs or when DNA damage, resulting from a bulky adduct or crosslinks, block progression of a high fidelity polymerase (for review, see [95]). The precise number of stalled forks to trigger the intra S-phase checkpoint is unknown [96]. Uncoupling of the helicase and DNA polymerase activity generates single strand gaps on both the leading and lagging strands. Checkpoint responses serve to maintain the stability of the replication fork in part by blocking the formation of toxic secondary DNA structures and replication fork reversal, degrading excessive histones, and inhibiting the firing of late replication forks so that replication can be resumed if stalled replication forks become permanently arrested [97]. In S phase checkpoint mutants, such as *rad53*, regressed replication forks, referred to as "chicken feet" structures, can be visualized [98]. Severe deficiencies can lead to mitotic catastrophe and subsequent lethality.

The extensive tracts of single-stranded DNA generated at stalled forks signal a checkpoint response. Similar, to checkpoint signaling at DSBs, the 9–1-1 complex is loaded and facilitates binding of Ddc2-Mec1. Rad18, which monoubiquitinates trimeric PCNA at K164 position at stalled replication forks, also monoubiquitinates the 9–1-1 complex leading to enhanced recruitment of Ddc2-Mec1 [99]. Checkpoint activation at stalled forks can also be facilitated by Elg1, which removes PCNA from stalled forks [100]. The Mrc1 (claspin) functions as an adaptor, analogous to Rad9, in the phosphorylation of Rad53; however, unlike Rad9, Mrc1 is associated with the replication forks [101]. Full checkpoint activation requires BLM homolog Sgs1 [102]. Rad53 phosphorylation in turn serves to promote histone degradation, inhibit late origin firing, and increase the levels of dNTPs. The inhibition of late origin firing maintains RPA and allows replication restart from other replication origins.

Deoxynucleotide levels (dNTPs) increase 7–8 fold after DNA damage by upregulating the activity of ribonucleotide reductase (Rnr) activity [103]. Upregulation of Rnr activity is achieved at the transcriptional, translational, and the posttranslational levels. At the transcriptional level, phosphorylated Rad53 activates Dun1 kinase, which deactivates Crt1 transcriptional repressor [63]. At the translational level, TRM9, which functions to methylate the uridine wobble base of tRNA-Arg (UCU) and tRNA-Glu (UUC), facilitates the translation of the Rnr1 transcript [104]; however, how checkpoint signaling enhances *TRM9* function is unclear. At the post-translational level, Dun1 inactivates the Sml1 protein inhibitor by phosphorylation [27]. Besides increasing the transcription of Rnr subunits, the Rnr inhibitor Sml1 and its paralog Dif1 are degraded, subsequently the Rnr subunits are shuttled to the cytoplasm where they form an active enzyme complex [64]. In addition to increasing the overall level of dNTPs, the Rnr3 large subunit forms an alternative ribonucleotide reductase complex that has relaxed dATP negative feedback regulation [105]. This ensures that adequate levels of dNTPs are available during times of unscheduled DNA synthesis.

While high levels of dNTPs facilitate replication fork progression [106]; abnormally high or low levels of dNTPs can promote genetic instability. High levels of dNTPs reduce the fidelity of polymerase epsilon [107]. Low levels of dNTPs correlate with hyper-recombination, as has been observed in *dun1* null mutants and in mec1 hypomorphs; these phenotypes can be suppressed by higher basal levels of dNTPs conferred by a *SML1* deletion [108]. These studies suggest that there is a range of dNTP concentrations that correlate with replication fork stability; however, the mechanisms by which higher dNTP levels decrease replication fork collapse are unclear.

In contrast to DSBs, where HR processes are facilitated, there are redundant mechanisms to prevent recombination at stalled forks; these mechanisms include disassembly of Rad51 filaments, helicases that abort recombination intermediates,

**79**

unclear.

**Figure 2.**

*replication bypass.*

**mechanisms**

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

and nucleases that degrade aberrant structures. PCNA is SUMOylated (SUMO-PCNA), facilitating the binding of the helicase Srs2, which suppresses recombination by disassembling Rad51 filaments (for review, see [109]. Recombination intermediates are aborted by the BLM ortholog Sgs1 helicase; indeed, the *sgs1 srs2* double mutants is not viable but viability is rescued when HR is defective [110]. Additional helicases, including Pif1 and Rrm3 can unwind and reverse "chicken feet" structures and reversed forks [66]. Nucleases, such as Exo1, function to degrade reversed forks, although excessive Exo1 activity can lead to replication fork collapse [62]. Finally, enzymes which cleave aberrant secondary structures, such as Yen1 and Mus4/Mus81, are inhibited or rendered less active by phosphorylation and Sumoylation (SUMO) [65]). Thus, there are multiple mechanisms that prevent

*The pathways of tolerating DNA damage at a stalled fork are shown by the arrows. The 5′ to 3′ polarity of the DNA is designated by an arrow. The Rrm3 and Pif1 helicases inhibit replication fork reversal, while the Pif1 helicase promotes template switching. Phosphorylated Rev1 binds to single-stranded DNA and promotes* 

aberrant structures from accumulating at replication blocks (**Figure 2**).

Generally, replication blocks that impede DNA polymerases can be bypassed by two mechanisms: 1) lesion bypass inserts a base opposite the replication block using error-prone or error-free translesion polymerases, and 2) template switch mechanisms [111] utilize recombination so that DNA polymerase bypasses DNA lesions on an undamaged template. Factors recruited to stalled forks would initially suggest that checkpoint signaling might favor lesion bypass by translesion polymerases. For example, Rad5 binding to stalled forks facilitates the recruitment of Rev1, even in the absence of DNA damage [112]. In addition, Rev1 is also phosphorylated by Mec1, which increases its affinity to ssDNA [26]. In vitro replication experiments have demonstrated that other error-prone polymerases can "jump start" replication, even without association of the replicative helicase [113]. However, there is no evidence that high levels of dNTPs would facilitate translesion synthesis mediated by error-prone polymerases, such as polζ [114], and the mechanism for jump start is

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

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

**Figure 2.**

*Saccharomyces*

mitotic catastrophe and subsequent lethality.

during times of unscheduled DNA synthesis.

major source of genetic instability [94]. Replication forks stall because of limiting amounts of dNTPs or when DNA damage, resulting from a bulky adduct or crosslinks, block progression of a high fidelity polymerase (for review, see [95]). The precise number of stalled forks to trigger the intra S-phase checkpoint is unknown [96]. Uncoupling of the helicase and DNA polymerase activity generates single strand gaps on both the leading and lagging strands. Checkpoint responses serve to maintain the stability of the replication fork in part by blocking the formation of toxic secondary DNA structures and replication fork reversal, degrading excessive histones, and inhibiting the firing of late replication forks so that replication can be resumed if stalled replication forks become permanently arrested [97]. In S phase checkpoint mutants, such as *rad53*, regressed replication forks, referred to as "chicken feet" structures, can be visualized [98]. Severe deficiencies can lead to

The extensive tracts of single-stranded DNA generated at stalled forks signal a checkpoint response. Similar, to checkpoint signaling at DSBs, the 9–1-1 complex is loaded and facilitates binding of Ddc2-Mec1. Rad18, which monoubiquitinates trimeric PCNA at K164 position at stalled replication forks, also monoubiquitinates the 9–1-1 complex leading to enhanced recruitment of Ddc2-Mec1 [99]. Checkpoint activation at stalled forks can also be facilitated by Elg1, which removes PCNA from stalled forks [100]. The Mrc1 (claspin) functions as an adaptor, analogous to Rad9, in the phosphorylation of Rad53; however, unlike Rad9, Mrc1 is associated with the replication forks [101]. Full checkpoint activation requires BLM homolog Sgs1 [102]. Rad53 phosphorylation in turn serves to promote histone degradation, inhibit late origin firing, and increase the levels of dNTPs. The inhibition of late origin firing maintains RPA and allows replication restart from other replication origins. Deoxynucleotide levels (dNTPs) increase 7–8 fold after DNA damage by upregulating the activity of ribonucleotide reductase (Rnr) activity [103]. Upregulation of Rnr activity is achieved at the transcriptional, translational, and the posttranslational levels. At the transcriptional level, phosphorylated Rad53 activates Dun1 kinase, which deactivates Crt1 transcriptional repressor [63]. At the translational level, TRM9, which functions to methylate the uridine wobble base of tRNA-Arg (UCU) and tRNA-Glu (UUC), facilitates the translation of the Rnr1 transcript [104]; however, how checkpoint signaling enhances *TRM9* function is unclear. At the post-translational level, Dun1 inactivates the Sml1 protein inhibitor by phosphorylation [27]. Besides increasing the transcription of Rnr subunits, the Rnr inhibitor Sml1 and its paralog Dif1 are degraded, subsequently the Rnr subunits are shuttled to the cytoplasm where they form an active enzyme complex [64]. In addition to increasing the overall level of dNTPs, the Rnr3 large subunit forms an alternative ribonucleotide reductase complex that has relaxed dATP negative feedback regulation [105]. This ensures that adequate levels of dNTPs are available

While high levels of dNTPs facilitate replication fork progression [106]; abnormally high or low levels of dNTPs can promote genetic instability. High levels of dNTPs reduce the fidelity of polymerase epsilon [107]. Low levels of dNTPs correlate with hyper-recombination, as has been observed in *dun1* null mutants and in mec1 hypomorphs; these phenotypes can be suppressed by higher basal levels of dNTPs conferred by a *SML1* deletion [108]. These studies suggest that there is a range of dNTP concentrations that correlate with replication fork stability; however, the mechanisms by which higher dNTP levels decrease replication fork

In contrast to DSBs, where HR processes are facilitated, there are redundant mechanisms to prevent recombination at stalled forks; these mechanisms include disassembly of Rad51 filaments, helicases that abort recombination intermediates,

**78**

collapse are unclear.

*The pathways of tolerating DNA damage at a stalled fork are shown by the arrows. The 5′ to 3′ polarity of the DNA is designated by an arrow. The Rrm3 and Pif1 helicases inhibit replication fork reversal, while the Pif1 helicase promotes template switching. Phosphorylated Rev1 binds to single-stranded DNA and promotes replication bypass.*

and nucleases that degrade aberrant structures. PCNA is SUMOylated (SUMO-PCNA), facilitating the binding of the helicase Srs2, which suppresses recombination by disassembling Rad51 filaments (for review, see [109]. Recombination intermediates are aborted by the BLM ortholog Sgs1 helicase; indeed, the *sgs1 srs2* double mutants is not viable but viability is rescued when HR is defective [110]. Additional helicases, including Pif1 and Rrm3 can unwind and reverse "chicken feet" structures and reversed forks [66]. Nucleases, such as Exo1, function to degrade reversed forks, although excessive Exo1 activity can lead to replication fork collapse [62]. Finally, enzymes which cleave aberrant secondary structures, such as Yen1 and Mus4/Mus81, are inhibited or rendered less active by phosphorylation and Sumoylation (SUMO) [65]). Thus, there are multiple mechanisms that prevent aberrant structures from accumulating at replication blocks (**Figure 2**).

Generally, replication blocks that impede DNA polymerases can be bypassed by two mechanisms: 1) lesion bypass inserts a base opposite the replication block using error-prone or error-free translesion polymerases, and 2) template switch mechanisms [111] utilize recombination so that DNA polymerase bypasses DNA lesions on an undamaged template. Factors recruited to stalled forks would initially suggest that checkpoint signaling might favor lesion bypass by translesion polymerases. For example, Rad5 binding to stalled forks facilitates the recruitment of Rev1, even in the absence of DNA damage [112]. In addition, Rev1 is also phosphorylated by Mec1, which increases its affinity to ssDNA [26]. In vitro replication experiments have demonstrated that other error-prone polymerases can "jump start" replication, even without association of the replicative helicase [113]. However, there is no evidence that high levels of dNTPs would facilitate translesion synthesis mediated by error-prone polymerases, such as polζ [114], and the mechanism for jump start is unclear.
