Checkpoint Control of DNA Repair in Yeast

*Michael Fasullo*

## **Abstract**

Budding yeast has been a model organism for understanding how DNA damage is repaired and how cells minimize genetic instability caused by arresting or delaying the cell cycle at well-defined checkpoints. However, many DNA damage insults are tolerated by mechanisms that can both be error-prone and error-free. The mechanisms that tolerate DNA damage and promote cell division are less well-understood. This review summarizes current information known about the checkpoint response to agents that elicit both the G2/M checkpoint and the intra-S phase checkpoint and how cells adapt to unrepaired DNA damage. Tolerance to particular bulky DNA adducts and radiomimetic agents are discussed, as well as possible mechanisms that may control phosphatases that deactivate phosphorylated proteins.

**Keywords:** DNA damage tolerance, checkpoint, budding yeast, phosphatase

### **1. Introduction**

DNA repair involves the recognition and excision of DNA damage followed by template-directed DNA synthesis using an undamaged strand (for reviews see [1]). Major repair mechanisms include base excision repair (BER, [2]), nucleotide excision repair (NER, [3]), and double-strand break (DSB) repair [4]; In budding yeast, homologous recombination (HR) is the preferred pathway for repair of DSBs. When the DNA replication apparatus bypasses DNA lesions on either the leading or lagging strand, single-strand gaps are created, and the resulting repair of gapped sister chromatids is referred to as postreplication repair (PRR); postreplication repair pathway involves DNA synthesis by low fidelity polymerases or template switch mechanisms. These studies have determined the identity of multiple components of these DNA repair pathways and demonstrated a remarkable conservation between both "simple" eukaryotes, such as budding yeast, and higher eukaryotes, including humans. Yeast studies, therefore, have a direct impact on understanding the molecular basis of inheritable DNA repair deficiencies in humans, many of which are associated with cancer (for review, see [5]).

One unifying theme of DNA repair is the redundancy of DNA repair mechanisms for specific DNA lesions. In budding yeast, the pathway choice may depend on the context of the DNA lesion, the stage of the cell cycle, and the ploidy of the strain. For example, DSBs can be repaired by non-homologous end joining (NHEJ), single-strand annealing (SSA), and HR [4]). HR is enhanced [6, 7] while NHEJ is suppressed in MATa/MATα diploid strains [8] where the HR repair can use an undamaged homolog as a repair template. Secondly, specific DNA base lesions, such as abasic sites can be repaired by both bases excision repair (BER) or nucleotide excision repair (NER, [2, 9, 10]). Thirdly, postreplication repair (PPR) pathways can be both error-prone and error-free [11]. While some DNA lesions are repaired by redundant mechanisms, others, such as inter-strand DNA cross-links that impede DNA polymerases, require components of multiple pathways, including NER, postreplication repair, and HR [12].

Cells adapt to unrepaired DNA lesions and rely on DNA damage tolerance mechanisms to maintain viability. For example, exposure to 150 J/m<sup>2</sup> UV, generates 3 x 104 cyclobutane pyrimidine dimers (CPD) per yeast cell, or approximately one UV-induced dimer per 400 bp of yeast DNA [13]. The pyrimidine-pyrimidone (6–4) photoproduct is also abundant but eightfold less present after exposure to UVB (280–320) [14]. The efficiency of CPD repair depends on the surrounding chromatin and whether the damaged strand is transcribed (for review, [15]). Since not all UV-induced damage is repaired within the period of a single cell cycle, cell viability depends on DNA damage tolerance and adaptation.

The purpose of this review is to summarize mechanisms by which checkpoint activation and DNA damage tolerance confer resistance for particular DNA lesions, and to summarize more recent data concerning complex carcinogen-associated lesions. The importance of this topic is underscored by observations that tolerance of DNA damage may reduce the efficacy of chemotherapeutic drugs, such as cisplatin, while increasing genetic instability. We present studies that suggest that DNA damage tolerance can be influenced by multiple factors, including the nutritional status of the cell and signaling from both the Target of Rapamycin (TOR) and the protein kinase A (PKA) pathways.

The orchestration of DNA repair pathways is especially critical when the replication fork progression is blocked by bulky DNA adducts [16]. Stalled replication forks can generate DNA secondary structures that trigger genome instability. While particular mechanisms to bypass such adducts, such as template switching and translesion DNA synthesis, may be error free, there is a risk that toxic recombination intermediates can either impede DNA replication progression or lead to replication fork collapse [17]. Replication-associated DNA breaks, in turn, may re-initiate replication on an undamaged chromosome or chromatid, referred to as break-induced replication (BIR, [18]). Chromosome breaks, if unrepaired, can be aberrantly rejoined forming dicentric chromosomes, leading to further breakage and instability, often referred to as the breakage-fusion-bridge cycle, a phenomena suggested to account for gross genome rearrangements in yeast [19] and in cancer cells [20, 21].

To suppress genetic instability and facilitate DNA repair, cell cycle checkpoints trigger arrest at defined stages in the cell cycle to ensure that DNA damage is repaired before the damage is replicated or inherited in the next cell cycle (for review, see [22]). These checkpoints are referred to as the G1-S checkpoint, the intra S checkpoint, and the G2/M checkpoint. In brief, phosphoinositide threekinase-related kinase (PI3K)-like kinases, referred to as apical or sensor kinases, initiate signaling after recruitment to DNA damage or stalled replication forks, checkpoint kinases, referred to as effector or downstream kinases, then amplify and transmit the checkpoint signal, and effectors that catalyze covalent protein modifications [23], resulting in activation or degradation of cellular targets. Besides ensuring that the cell cycle is delayed so that adequate time is available for DNA repair [24], effectors also modify DNA repair proteins [25, 26]), upregulate the synthesis of deoxynucleotides (dNTPs, [27]), regulate transport of tRNA from the nucleus to and from the cytoplasm, trigger autophagy [28], regulate histone levels [29], and cross-talk with other stress-induced pathways to ensure survival [30]. The totality of the response is generally referred to as the DNA

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

insult and after DNA repair is completed.

**2. Checkpoint activation initiated by DSBs**

still induced [39] and Rad55 is phosphorylated [38] .

damage response (DDR) [22]. Checkpoint recovery occurs once the DNA damage is repaired or the replication block has been circumvented [31]; however, adaptation to persistent DNA lesions, such as DSBs, can also occur. In either case, the phosphorylated and activated checkpoint proteins are either dephosphorylated or degraded and subsequently rendered inactive. Depending on the DNA damage and time period of exposure, peak activation occurs within 2–4 hours after acute DNA damage exposure [32], with simultaneous upregulation of dNTP levels and DNA damage-inducible genes. Repression of late origins of replication, inhibition of replication, and prevention of anaphase can last for additional hours [33], while adaptation can be observed after 12 hrs. The presence of DNA repair foci, such as Rad51, can thus last hours after the initiation of the DNA damage insult. Thus, aspects of the DNA damage response can persist hours after the initial genomic

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

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].

*Saccharomyces*

ates 3 x 104

NER, postreplication repair, and HR [12].

protein kinase A (PKA) pathways.

as abasic sites can be repaired by both bases excision repair (BER) or nucleotide excision repair (NER, [2, 9, 10]). Thirdly, postreplication repair (PPR) pathways can be both error-prone and error-free [11]. While some DNA lesions are repaired by redundant mechanisms, others, such as inter-strand DNA cross-links that impede DNA polymerases, require components of multiple pathways, including

Cells adapt to unrepaired DNA lesions and rely on DNA damage tolerance

one UV-induced dimer per 400 bp of yeast DNA [13]. The pyrimidine-pyrimidone (6–4) photoproduct is also abundant but eightfold less present after exposure to UVB (280–320) [14]. The efficiency of CPD repair depends on the surrounding chromatin and whether the damaged strand is transcribed (for review, [15]). Since not all UV-induced damage is repaired within the period of a single cell cycle, cell

The purpose of this review is to summarize mechanisms by which checkpoint activation and DNA damage tolerance confer resistance for particular DNA lesions, and to summarize more recent data concerning complex carcinogen-associated lesions. The importance of this topic is underscored by observations that tolerance of DNA damage may reduce the efficacy of chemotherapeutic drugs, such as cisplatin, while increasing genetic instability. We present studies that suggest that DNA damage tolerance can be influenced by multiple factors, including the nutritional status of the cell and signaling from both the Target of Rapamycin (TOR) and the

The orchestration of DNA repair pathways is especially critical when the replication fork progression is blocked by bulky DNA adducts [16]. Stalled replication forks can generate DNA secondary structures that trigger genome instability. While particular mechanisms to bypass such adducts, such as template switching and translesion DNA synthesis, may be error free, there is a risk that toxic recombination intermediates can either impede DNA replication progression or lead to replication fork collapse [17]. Replication-associated DNA breaks, in turn, may re-initiate replication on an undamaged chromosome or chromatid, referred to as break-induced replication (BIR, [18]). Chromosome breaks, if unrepaired, can be aberrantly rejoined forming dicentric chromosomes, leading to further breakage and instability, often referred to as the breakage-fusion-bridge cycle, a phenomena suggested to account for gross genome rearrangements in yeast [19] and in cancer

To suppress genetic instability and facilitate DNA repair, cell cycle checkpoints

trigger arrest at defined stages in the cell cycle to ensure that DNA damage is repaired before the damage is replicated or inherited in the next cell cycle (for review, see [22]). These checkpoints are referred to as the G1-S checkpoint, the intra S checkpoint, and the G2/M checkpoint. In brief, phosphoinositide threekinase-related kinase (PI3K)-like kinases, referred to as apical or sensor kinases, initiate signaling after recruitment to DNA damage or stalled replication forks, checkpoint kinases, referred to as effector or downstream kinases, then amplify and transmit the checkpoint signal, and effectors that catalyze covalent protein modifications [23], resulting in activation or degradation of cellular targets. Besides ensuring that the cell cycle is delayed so that adequate time is available for DNA repair [24], effectors also modify DNA repair proteins [25, 26]), upregulate the synthesis of deoxynucleotides (dNTPs, [27]), regulate transport of tRNA from the nucleus to and from the cytoplasm, trigger autophagy [28], regulate histone levels [29], and cross-talk with other stress-induced pathways to ensure survival [30]. The totality of the response is generally referred to as the DNA

cyclobutane pyrimidine dimers (CPD) per yeast cell, or approximately

UV, gener-

mechanisms to maintain viability. For example, exposure to 150 J/m<sup>2</sup>

viability depends on DNA damage tolerance and adaptation.

**72**

cells [20, 21].

damage response (DDR) [22]. Checkpoint recovery occurs once the DNA damage is repaired or the replication block has been circumvented [31]; however, adaptation to persistent DNA lesions, such as DSBs, can also occur. In either case, the phosphorylated and activated checkpoint proteins are either dephosphorylated or degraded and subsequently rendered inactive. Depending on the DNA damage and time period of exposure, peak activation occurs within 2–4 hours after acute DNA damage exposure [32], with simultaneous upregulation of dNTP levels and DNA damage-inducible genes. Repression of late origins of replication, inhibition of replication, and prevention of anaphase can last for additional hours [33], while adaptation can be observed after 12 hrs. The presence of DNA repair foci, such as Rad51, can thus last hours after the initiation of the DNA damage insult. Thus, aspects of the DNA damage response can persist hours after the initial genomic insult and after DNA repair is completed.
