**3. Roles of RecQ helicase in telomere maintenance**

RecQ helicase is conserved from *E. coli.* to human and play a critical role in genome stability (Bernstein, Gangloff, and Rothstein 2010). Werner Syndrome (WS) is a premature aging syndrome resulting from loss of function of one of the human RecQ helicase WRN. The roles of *S. cerevisiae* RecQ helicase Sgs1 in homologous recombination are well studied. RecQ helicase is also involved in telomere maintenance especially at dysfunctional telomere. In this section, roles of RecQ helicase in telomere maintenance will be discussed. Functional interaction between RecQ helicase and POT1 in *S. pombe* and in human will be also discussed.

#### **3.1 Roles of RecQ helicase in DNA repair**

*S. cerevisiae* RecQ helicase Sgs1 is involved in several steps in HR (Ashton and Hickson 2010). As discussed above, Sgs1 is involved in the resection of DSB ends. Genetic and in vitro studies also suggest that Sgs1 inhibits unscheduled recombinogenic events, but promotes the resolution of recombination intermediates. Strains deleted for *SGS1* display hyperrecombination phenotype, but are defective in DNA damage-induced heteroallilic

Fig. 2. Model for DNA end-processing at dysfunctional telomere. 3' single-stranded

**2.4 Proteins involved in DNA end-processing in** *S. cerevisiae cdc13-1* **cells** 

unknown nuclease, but not nuclease activity depending on MRN-Dna2. 3' single-stranded overhangs are produced by Pif1 or Exo1 in *S. cerevisiae cdc13-1* cells (Right). Unknown

*S. cerevisiae* Cdc13 binds telomeric single-stranded DNA (Garvik, Carson, and Hartwell 1995). *cdc13-1* temperature sensitive mutant is used to study proteins that are involved in the resection at uncapped telomeres (Lydall 2009). These studies revealed that the singlestranded DNA at telomeres in *cdc13-1* mutants resembles a DSB end. However, there are some differences between these ends (Fig. 2). In *cdc13-1* mutants at high temperature, Pif1 helicase and Exo1 are redundantly involved in the resection of uncapped telomere (Dewar and Lydall 2010). It remains unclear how Pif1 contribute to the resection. As Pif1 has no nuclease activity, involvement of the unknown nuclease is suggested to cleave singlestranded DNA unwound by Pif1 helicase. Sgs1 also contributes to resection of telomeres in *cdc13-1* mutants (Ngo and Lydall 2010). However, unlike *pif1 exo1* double mutant, resection of telomeres in *cdc13-1* mutant background occurs in *sgs1 exo1* double mutant, demonstrating that Pif1 and Exo1 play major roles in the resection of uncapped telomere

RecQ helicase is conserved from *E. coli.* to human and play a critical role in genome stability (Bernstein, Gangloff, and Rothstein 2010). Werner Syndrome (WS) is a premature aging syndrome resulting from loss of function of one of the human RecQ helicase WRN. The roles of *S. cerevisiae* RecQ helicase Sgs1 in homologous recombination are well studied. RecQ helicase is also involved in telomere maintenance especially at dysfunctional telomere. In this section, roles of RecQ helicase in telomere maintenance will be discussed. Functional interaction between RecQ helicase and POT1 in *S. pombe* and in human will be also

*S. cerevisiae* RecQ helicase Sgs1 is involved in several steps in HR (Ashton and Hickson 2010). As discussed above, Sgs1 is involved in the resection of DSB ends. Genetic and in vitro studies also suggest that Sgs1 inhibits unscheduled recombinogenic events, but promotes the resolution of recombination intermediates. Strains deleted for *SGS1* display hyperrecombination phenotype, but are defective in DNA damage-induced heteroallilic

Δ

cells (Left). Ku inhibits

overhangs are produced by MRN and Dna2 in *S. pombe taz1*

nuclease is suggested to function together with Pif1 helicase.

**3. Roles of RecQ helicase in telomere maintenance** 

**3.1 Roles of RecQ helicase in DNA repair** 

at high temperature.

discussed.

recombination (Watt et al. 1996) (Onoda et al. 2001). *S. cerevisiae* Sgs1 and Top3 migrate and disentangle a double Holliday junction (dHJ) to produce non-crossover recombination products in vitro (Cejka et al. 2010). This activity is also detected in human RecQ helicase BLM and human topoisomerase IIIa (Wu and Hickson 2003). Mutant of *S. pombe* RecQ helicase *rqh1* is sensitive to DNA damage and has high frequency of recombination under normal growth conditions and following DNA damage, suggesting that Rqh1 is also involved in HR repair both positively and negatively (Murray et al. 1997) (Stewart et al. 1997) (Doe et al. 2000) (Caspari, Murray, and Carr 2002).

#### **3.2 Roles of RecQ helicase in telomere maintenance in** *S. cerevisiae*

As mutation of *S. cerevisiae SGS1* does not affect telomere length, Sgs1 has no apparent role in telomere maintenance in the presence of telomerase activity (Watt et al. 1996). However, the double mutant between telomerase RNA component *TLC1* and *SGS1* shorten telomeres at an increased rate per population doubling and Sgs1 affects telomere-telomere recombination in the absence of telomerase, demonstrating that Sgs1 plays roles at telomere in the absence of telomerase activity (Johnson et al. 2001) (Cohen and Sinclair 2001) (Huang et al. 2001). Xshaped structures are accumulated at telomeres in senescing *tlc1 sgs1* double mutants and these structures are suggested to be the recombination intermediates related to hemicatenanes. This result suggests that Sgs1 is required for the efficient resolution of telomere recombination intermediates in the absence of telomerase (Lee et al. 2007; Chavez, Tsou, and Johnson 2009).

#### **3.3 Roles of RecQ helicase in telomere maintenance in mammals**

Human RecQ helicase WRN binds to telomere in S phase in primary human IMR90 fibroblasts and is required for efficient replication of the G-rich telomeric DNA strand, suggesting that WRN is required for replication of telomeric DNA in telomerase-negative primary human fibroblasts (Crabbe et al. 2004). In Werner syndrome (WS) cells, replicationassociated telomere loss results in the chromosome fusions, causing genomic instability (Crabbe et al. 2007). The life span of normal human skin fibroblasts derived from WS patients can be extended by expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) (Wyllie et al. 2000; Ouellette et al. 2000). These facts demonstrate that dysfunctional telomere is a major determinant of the premature aging syndrome and WRN plays important role at dysfunctional telomere and telomerase activity can suppress the defect in WRN deficient cells. Consistently, Wrn-deficient mouse, which has telomerase activity, has no disease phenotype, but telomerase-Wrn double null mouse elicits a Wernerlike premature aging syndrome (Chang et al. 2004). Telomere sister chromatid exchange (T-SEC) increases in cells from telomerase-Wrn double null mouse, suggesting that WRN are required to repress inappropriate telomere recombination (Laud et al. 2005) (Multani and Chang 2007). Human WRN and other RecQ helicase BLM co-localizes with telomere in human cancer cells that lack telomerase, ALT cells (Johnson et al. 2001; Opresko et al. 2004; Lillard-Wetherell et al. 2004). As telomeres in ALT cells are maintained by HR, human WRN and BLM are suggested to be involved in the recombination at telomere in ALT cells. Possible roles of WRN in telomere maintenance will be discussed in the next section.

#### **3.4 Functional interaction between RecQ helicase and POT1 in** *S. pombe* **and in human**

Pot1 is conserved from *S. pombe* to human and binds to single-stranded telomeric DNA sequence specifically (Baumann and Cech 2001). Deletion of *S. pombe pot1* causes rapid

Roles of DNA Repair Proteins in Telomere Maintenance 603

inhibition of WRN and POT1 also lender human cells sensitive to anti-microtubule drug vinblastine, implying the functional conservation between human POT1 and WRN and *S. pombe* Pot1 and Rqh1(Takahashi et al. 2011). The other double knockdown experiments of WRN and POT1 in human cells show that human POT1 is required for efficient telomere Crich strand replication in the absence of WRN (Arnoult et al. 2009). The functional interaction between human POT1 and RecQ helicase WRN is also suggested by in vitro experiment. Purified human POT1 binds to WRN and POT1 binding on telomeric DNA regulates the unwinding activity of WRN (Opresko et al. 2005; Sowd, Lei, and Opresko 2008; Opresko, Sowd, and Wang 2009). Based on these and other data, several possible roles of WRN at telomere are suggested (Rossi, Ghosh, and Bohr 2010) (Fig. 3). Telomere is capped by telomere binding proteins called shelterin and the chromosome end is protected through strand invasion of the duplex telomeric repeat by the 3' single-stranded overhangs, which is called t-loop (Palm and de Lange 2008). As WRN acts to release the 3' invading tail from a telomeric D loop in vitro, WRN may be involved in the regulation of the t-loop (Opresko et al. 2004). Single-stranded overhangs can fold into G-quadruplex DNA, which may inhibit DNA polymerase and telomerase at telomere (Zaug, Podell, and Cech 2005). Therefore, WRN may disrupt telomeric G-quadruplex with POT1 to facilitate DNA replication and/or

Replication protein A (RPA) is a heterotrimeric single-stranded non-specific DNA-binding protein consisting of a large (70 kDa), middle (32 kDa) and small (14 kDa) subunit. RPA is conserved from yeast to human and is essential for DNA replication, repair, and recombination (Binz, Sheehan, and Wold 2004). The large subunits of RPA in human, *S, cerevisiae* and *S. pombe* are named as RPA70, Rfa1 and Rad11, respectively. RPA is involved in HR repair by binding the single-stranded DNA generated by DNA end-processing at DSB ends. Single-stranded DNA is also produced at telomere. But RPA is suggested to be excluded from single-stranded telomere overhangs because it will lead to DNA damage checkpoint activation and cell cycle arrest. However, genetic evidences suggest the role of RPA in telomere maintenance. In this section, possible roles of RPA in telomere maintenance will be discussed. The functional relationship between RPA, RecQ helicase, and Taz1 will be

Mutations in *S, cerevisiare rfa1* lender cells to sensitive to DNA damage and affect recombination efficiency, suggesting the involvement of RPA in recombination and repair processes (Smith and Rothstein 1995; Firmenich, Elias-Arnanz, and Berg 1995; Umezu et al. 1998). *S. pombe rad11* mutants are also sensitive to DNA damage and *rad11-D223Y* mutant is epitatic to *rad50* mutant, suggesting that RPA is involved in the HR repair (Parker et al. 1997; Ono et al. 2003). The roles of RPA in HR repair is well studied by in vitro system using *S. pombe* proteins (Kurokawa et al. 2008; Murayama et al. 2008). These in vitro and other genetic studies suggest that RPA binds to the single-stranded DNA generated by processing at DSB end. Then Rad22 (the *S. pombe* Rad52 homolog) helps Rad51 to displace RPA from single-stranded DNA. RPA bound to the single-stranded DNA recruits DNA damage checkpoint proteins to the DSB site to activate DNA damage

telomere elongation at telomeres.

**4.1 Roles of RPA in DNA repair** 

checkpoint (Zou and Elledge 2003).

also discussed.

**4. Roles of RPA in telomere maintenance** 

telomere loss and chromosome circularization and this circularization is mediated by single strand annealing (SSA) (Wang and Baumann 2008). In *S. cerevisiae*, Rad52, Rad1/Rad10 nuclease, RPA, Srs2 helicase, and Sgs1 are involved in SSA (Fishman-Lobell and Haber 1992) (Ivanov and Haber 1995) (Ivanov et al. 1996) (Paques and Haber 1997), (Sugawara, Ira, and Haber 2000; Umezu et al. 1998) (Zhu et al. 2008). Consistently, the double mutants between *S. pombe* homologue of these proteins and *pot1* are synthetically lethal (Wang and Baumann 2008). *S. pombe* telomerase disruptant can survive either by maintaining telomere by HR or chromosome circularization(Nakamura, Cooper, and Cech 1998). In contrast, *pot1* disruptant survives only by chromosome circularization (Baumann and Cech 2001). One possible explanation is that Pot1 is required for prevention of rapid telomere loss, which would lead chromosome circularization dominantly. Recently our group has reported that the double mutant between *rqh1-hd* (helicase dead point mutant) and *pot1* is not synthetically lethal (Takahashi et al. 2011). The chromosome ends of the *pot1 rqh1-hd* double mutant are maintained by HR. There are several possible explanations for this. First, helicase dead Rqh1 may bind to the chromosome ends in *pot1* disruptant to inhibit rapid telomere loss, allowing cells to maintain chromosome ends by HR. Second, helicase activity of the Rqh1 may be involved in the rapid telomere loss in the *pot1* disruptant, because *S. cerevisiae* RecQ helicase is involved in the processing of telomere ends. This will also allow cells to maintain chromosome ends by HR. Third, helicase activity of the Rqh1 may be required for the suppression of recombination at telomere. This will also allow cells to maintain chromosome ends by HR. The exact role of the helicase dead Rqh1 in pot1 disruptant remains unclear. Interestingly, *pot1 rqh1-hd* double mutant is sensitive to anti-microtubule drug thiabendazole (TBZ) (Takahashi et al. 2011). The *pot1 rqh1-hd* double mutant has recombination intermediates even in the M phase at the chromosome ends. This physical link between the sister chromatids in M phase will inhibit chromosome segregation, especially in the presence of TBZ, which would lender cells sensitive to TBZ. Interestingly, concomitant

Fig. 3. WRN activities on a telomeric D-loop structure (A) and on a lagging strand telomere (B) during S phase. **A**. The model shows that WRN helicase releases the invading strand during S phase. **B**. WRN resolves G-quartet (G) formed on the lagging telomeric DNA.

telomere loss and chromosome circularization and this circularization is mediated by single strand annealing (SSA) (Wang and Baumann 2008). In *S. cerevisiae*, Rad52, Rad1/Rad10 nuclease, RPA, Srs2 helicase, and Sgs1 are involved in SSA (Fishman-Lobell and Haber 1992) (Ivanov and Haber 1995) (Ivanov et al. 1996) (Paques and Haber 1997), (Sugawara, Ira, and Haber 2000; Umezu et al. 1998) (Zhu et al. 2008). Consistently, the double mutants between *S. pombe* homologue of these proteins and *pot1* are synthetically lethal (Wang and Baumann 2008). *S. pombe* telomerase disruptant can survive either by maintaining telomere by HR or chromosome circularization(Nakamura, Cooper, and Cech 1998). In contrast, *pot1* disruptant survives only by chromosome circularization (Baumann and Cech 2001). One possible explanation is that Pot1 is required for prevention of rapid telomere loss, which would lead chromosome circularization dominantly. Recently our group has reported that the double mutant between *rqh1-hd* (helicase dead point mutant) and *pot1* is not synthetically lethal (Takahashi et al. 2011). The chromosome ends of the *pot1 rqh1-hd* double mutant are maintained by HR. There are several possible explanations for this. First, helicase dead Rqh1 may bind to the chromosome ends in *pot1* disruptant to inhibit rapid telomere loss, allowing cells to maintain chromosome ends by HR. Second, helicase activity of the Rqh1 may be involved in the rapid telomere loss in the *pot1* disruptant, because *S. cerevisiae* RecQ helicase is involved in the processing of telomere ends. This will also allow cells to maintain chromosome ends by HR. Third, helicase activity of the Rqh1 may be required for the suppression of recombination at telomere. This will also allow cells to maintain chromosome ends by HR. The exact role of the helicase dead Rqh1 in pot1 disruptant remains unclear. Interestingly, *pot1 rqh1-hd* double mutant is sensitive to anti-microtubule drug thiabendazole (TBZ) (Takahashi et al. 2011). The *pot1 rqh1-hd* double mutant has recombination intermediates even in the M phase at the chromosome ends. This physical link between the sister chromatids in M phase will inhibit chromosome segregation, especially in the presence of TBZ, which would lender cells sensitive to TBZ. Interestingly, concomitant

Fig. 3. WRN activities on a telomeric D-loop structure (A) and on a lagging strand telomere (B) during S phase. **A**. The model shows that WRN helicase releases the invading strand during S phase. **B**. WRN resolves G-quartet (G) formed on the lagging telomeric DNA.

inhibition of WRN and POT1 also lender human cells sensitive to anti-microtubule drug vinblastine, implying the functional conservation between human POT1 and WRN and *S. pombe* Pot1 and Rqh1(Takahashi et al. 2011). The other double knockdown experiments of WRN and POT1 in human cells show that human POT1 is required for efficient telomere Crich strand replication in the absence of WRN (Arnoult et al. 2009). The functional interaction between human POT1 and RecQ helicase WRN is also suggested by in vitro experiment. Purified human POT1 binds to WRN and POT1 binding on telomeric DNA regulates the unwinding activity of WRN (Opresko et al. 2005; Sowd, Lei, and Opresko 2008; Opresko, Sowd, and Wang 2009). Based on these and other data, several possible roles of WRN at telomere are suggested (Rossi, Ghosh, and Bohr 2010) (Fig. 3). Telomere is capped by telomere binding proteins called shelterin and the chromosome end is protected through strand invasion of the duplex telomeric repeat by the 3' single-stranded overhangs, which is called t-loop (Palm and de Lange 2008). As WRN acts to release the 3' invading tail from a telomeric D loop in vitro, WRN may be involved in the regulation of the t-loop (Opresko et al. 2004). Single-stranded overhangs can fold into G-quadruplex DNA, which may inhibit DNA polymerase and telomerase at telomere (Zaug, Podell, and Cech 2005). Therefore, WRN may disrupt telomeric G-quadruplex with POT1 to facilitate DNA replication and/or telomere elongation at telomeres.

### **4. Roles of RPA in telomere maintenance**

Replication protein A (RPA) is a heterotrimeric single-stranded non-specific DNA-binding protein consisting of a large (70 kDa), middle (32 kDa) and small (14 kDa) subunit. RPA is conserved from yeast to human and is essential for DNA replication, repair, and recombination (Binz, Sheehan, and Wold 2004). The large subunits of RPA in human, *S, cerevisiae* and *S. pombe* are named as RPA70, Rfa1 and Rad11, respectively. RPA is involved in HR repair by binding the single-stranded DNA generated by DNA end-processing at DSB ends. Single-stranded DNA is also produced at telomere. But RPA is suggested to be excluded from single-stranded telomere overhangs because it will lead to DNA damage checkpoint activation and cell cycle arrest. However, genetic evidences suggest the role of RPA in telomere maintenance. In this section, possible roles of RPA in telomere maintenance will be discussed. The functional relationship between RPA, RecQ helicase, and Taz1 will be also discussed.

#### **4.1 Roles of RPA in DNA repair**

Mutations in *S, cerevisiare rfa1* lender cells to sensitive to DNA damage and affect recombination efficiency, suggesting the involvement of RPA in recombination and repair processes (Smith and Rothstein 1995; Firmenich, Elias-Arnanz, and Berg 1995; Umezu et al. 1998). *S. pombe rad11* mutants are also sensitive to DNA damage and *rad11-D223Y* mutant is epitatic to *rad50* mutant, suggesting that RPA is involved in the HR repair (Parker et al. 1997; Ono et al. 2003). The roles of RPA in HR repair is well studied by in vitro system using *S. pombe* proteins (Kurokawa et al. 2008; Murayama et al. 2008). These in vitro and other genetic studies suggest that RPA binds to the single-stranded DNA generated by processing at DSB end. Then Rad22 (the *S. pombe* Rad52 homolog) helps Rad51 to displace RPA from single-stranded DNA. RPA bound to the single-stranded DNA recruits DNA damage checkpoint proteins to the DSB site to activate DNA damage checkpoint (Zou and Elledge 2003).

Roles of DNA Repair Proteins in Telomere Maintenance 605

*S. pombe taz1 rad11-D223Y* double mutant lose telomere very rapidly, demonstrating that Taz1 and RPA collaborate to maintain telomere (Kibe et al. 2007). This rapid telomere loss can be suppressed by overexpression of Pot1. One possible explanation for this data is that Taz1 and RPA are required for the function of Pot1 at telomere and overexpression of Pot1 can rescue this defect. The rapid telomere loss of *taz1 rad11-D223Y* double mutant can be also suppressed by deletion of *rqh1*. Sgs1 is involved in the processing of telomere ends in *S. cerevisiae*. Similarly, *S. pombe* Rqh1 may be involved in the rapid telomere loss, possible by degradation of C-rich strand in *taz1 rad11-D223Y* double mutant (Fig. 5). The other functional relationship between Taz1 and Rqh1 is reported by Cooper group. *taz1* disruptant is sensitive to low temperature (Miller and Cooper 2003). Telomere entanglement is suggested to be a reason for this cold sensitivity. They found that unsumoylated Rqh1 mutant can suppress this cold sensitivity (Rog et al. 2009). Trt1 is a catalitic subunit of telomerase in *S. pombe*. *trt1* single mutant loses telomeric DNA gradually (Nakamura, Cooper, and Cech 1998). In contrast, *taz1 trt1* double mutant lose telomere very rapidly (Miller, Rog, and Cooper 2006). The replication fork stalling at the telomeres and resultant DSB is suggested to be a season for the rapid telomere loss in *taz1 trt1* double mutant. Unsumoylated Rqh1 mutant can also suppress this rapid telomere loss. Based on these data, they propose that sumoylated Rqh1 promotes telomere breakage and entanglement in *taz1* disruptant. This data demonstrate that the activity of Rqh1 at telomere is regulated to protect telomere. However, it remains unclear how Rqh1 and other DNA repair proteins are regulated at telomere. The functional interactions between human TRF1/TRF2 (*S. pombe*

Fig. 5. The model shows that *S. pombe* Taz1 and RPA are required for prevent rapid telomere loss. In *taz1 rad11-D223Y* double mutant, Pot1 can not function properly and Rqh1 and

possibly Dna2 resects telomere ends, which causes rapid telomere loss.

**4.3 Functional interaction between** *S. pombe* **Taz1, RPA and RecQ helicase** 

#### **4.2 Roles of RPA in telomere maintenance**

Telomere ends have single-strand overhangs, which may serve substrates for RPA. However, it is belieaved that RPA is excluded from telomere to suppress DNA damage checkpoint activation at telomere. Indeed, binding of human and mouse POT1 to telomeric ssDNA inhibits the localization of RPA to telomeres (Barrientos et al. 2008) (Gong and de Lange 2010). However, there are several genetic evidences suggesting that RPA is involved in telomere maintenance. Mutation of *S. cerevisiae RFA1* gene, *rfa1-D228Y* in *Yku70* mutant background causes telomere shortening, demonstration that RPA is required for telomere length regulation at dysfunctional telomere (Smith, Zou, and Rothstein 2000). Moreover, certain mutant alleles of *RFA2* gene, encoding the middle subunit of RPA, in wild-type background causes telomere shortening, demonstration that RPA is required for telomere length regulation (Mallory et al. 2003). In addition, *S. cerevisiae* RPA binds to telomere especially in S phase and cells expressing truncated Rfa2 show impaired binding of the Est1, a component of telomerase (Schramke et al. 2004). Based on these data, they proposed that RPA activates telomerase by loading Est1 onto telomeres during S phase. *S. pombe rad11- D223Y* mutant, which corresponds to the *S.cerevisiae rfa1-D228Y* mutant, has short telomere in wild-type background. Moreover, *S. pombe* RPA binds to telomere especially in S phase (Ono et al. 2003; Moser et al. 2009). A genome-wide screen for *S. pombe* deletion mutants shows that deletion of *ssb3*, the small subunit of RPA, affects telomere length(Liu et al. 2010). These facts suggest that RPA plays important role in telomere maintenance in both *S. cerevisiae* and *S. pombe*. Human RPA is also enriched at telomere during S phase, possibly due to exposure of single-stranded DNA during telomere replication (Verdun and Karlseder 2006). The aspartic acid at position 223 in *S. pombe* Rad11 is important for telomere length regulation, which corresponds to the position 227 in human RPA70 (Ono et al. 2003). Similarly, expression of RPA70-D227Y mutant protein in human fibrosarcoma HT1080 cells causes telomere shortening, suggesting that human RPA also plays role in telomere length regulation (Kobayashi et al. 2010). Possible role of RPA at telomere is the regulation of the processing of telomere ends by controlling accessibility of DNA repair proteins and/or Pot1 to single-stranded overhang (Fig. 4).

Fig. 4. The model shows that *S. pombe* RPA regulates the localizations and/or activities of proteins involved in the telomere maintenance. RPA may regulate Dna2 and/or Rqh1 during S phase.

Telomere ends have single-strand overhangs, which may serve substrates for RPA. However, it is belieaved that RPA is excluded from telomere to suppress DNA damage checkpoint activation at telomere. Indeed, binding of human and mouse POT1 to telomeric ssDNA inhibits the localization of RPA to telomeres (Barrientos et al. 2008) (Gong and de Lange 2010). However, there are several genetic evidences suggesting that RPA is involved in telomere maintenance. Mutation of *S. cerevisiae RFA1* gene, *rfa1-D228Y* in *Yku70* mutant background causes telomere shortening, demonstration that RPA is required for telomere length regulation at dysfunctional telomere (Smith, Zou, and Rothstein 2000). Moreover, certain mutant alleles of *RFA2* gene, encoding the middle subunit of RPA, in wild-type background causes telomere shortening, demonstration that RPA is required for telomere length regulation (Mallory et al. 2003). In addition, *S. cerevisiae* RPA binds to telomere especially in S phase and cells expressing truncated Rfa2 show impaired binding of the Est1, a component of telomerase (Schramke et al. 2004). Based on these data, they proposed that RPA activates telomerase by loading Est1 onto telomeres during S phase. *S. pombe rad11- D223Y* mutant, which corresponds to the *S.cerevisiae rfa1-D228Y* mutant, has short telomere in wild-type background. Moreover, *S. pombe* RPA binds to telomere especially in S phase (Ono et al. 2003; Moser et al. 2009). A genome-wide screen for *S. pombe* deletion mutants shows that deletion of *ssb3*, the small subunit of RPA, affects telomere length(Liu et al. 2010). These facts suggest that RPA plays important role in telomere maintenance in both *S. cerevisiae* and *S. pombe*. Human RPA is also enriched at telomere during S phase, possibly due to exposure of single-stranded DNA during telomere replication (Verdun and Karlseder 2006). The aspartic acid at position 223 in *S. pombe* Rad11 is important for telomere length regulation, which corresponds to the position 227 in human RPA70 (Ono et al. 2003). Similarly, expression of RPA70-D227Y mutant protein in human fibrosarcoma HT1080 cells causes telomere shortening, suggesting that human RPA also plays role in telomere length regulation (Kobayashi et al. 2010). Possible role of RPA at telomere is the regulation of the processing of telomere ends by controlling accessibility of DNA repair proteins and/or Pot1

Fig. 4. The model shows that *S. pombe* RPA regulates the localizations and/or activities of proteins involved in the telomere maintenance. RPA may regulate Dna2 and/or Rqh1

**4.2 Roles of RPA in telomere maintenance** 

to single-stranded overhang (Fig. 4).

during S phase.

#### **4.3 Functional interaction between** *S. pombe* **Taz1, RPA and RecQ helicase**

*S. pombe taz1 rad11-D223Y* double mutant lose telomere very rapidly, demonstrating that Taz1 and RPA collaborate to maintain telomere (Kibe et al. 2007). This rapid telomere loss can be suppressed by overexpression of Pot1. One possible explanation for this data is that Taz1 and RPA are required for the function of Pot1 at telomere and overexpression of Pot1 can rescue this defect. The rapid telomere loss of *taz1 rad11-D223Y* double mutant can be also suppressed by deletion of *rqh1*. Sgs1 is involved in the processing of telomere ends in *S. cerevisiae*. Similarly, *S. pombe* Rqh1 may be involved in the rapid telomere loss, possible by degradation of C-rich strand in *taz1 rad11-D223Y* double mutant (Fig. 5). The other functional relationship between Taz1 and Rqh1 is reported by Cooper group. *taz1* disruptant is sensitive to low temperature (Miller and Cooper 2003). Telomere entanglement is suggested to be a reason for this cold sensitivity. They found that unsumoylated Rqh1 mutant can suppress this cold sensitivity (Rog et al. 2009). Trt1 is a catalitic subunit of telomerase in *S. pombe*. *trt1* single mutant loses telomeric DNA gradually (Nakamura, Cooper, and Cech 1998). In contrast, *taz1 trt1* double mutant lose telomere very rapidly (Miller, Rog, and Cooper 2006). The replication fork stalling at the telomeres and resultant DSB is suggested to be a season for the rapid telomere loss in *taz1 trt1* double mutant. Unsumoylated Rqh1 mutant can also suppress this rapid telomere loss. Based on these data, they propose that sumoylated Rqh1 promotes telomere breakage and entanglement in *taz1* disruptant. This data demonstrate that the activity of Rqh1 at telomere is regulated to protect telomere. However, it remains unclear how Rqh1 and other DNA repair proteins are regulated at telomere. The functional interactions between human TRF1/TRF2 (*S. pombe*

Fig. 5. The model shows that *S. pombe* Taz1 and RPA are required for prevent rapid telomere loss. In *taz1 rad11-D223Y* double mutant, Pot1 can not function properly and Rqh1 and possibly Dna2 resects telomere ends, which causes rapid telomere loss.

Roles of DNA Repair Proteins in Telomere Maintenance 607

Bernstein, K. A., S. Gangloff, and R. Rothstein. 2010. The RecQ DNA helicases in DNA

Binz, S. K., A. M. Sheehan, and M. S. Wold. 2004. Replication protein A phosphorylation and the cellular response to DNA damage. *DNA Repair (Amst)* 3 (8-9):1015-24. Bonetti, D., M. Martina, M. Clerici, G. Lucchini, and M. P. Longhese. 2009. Multiple

Carneiro, T., L. Khair, C. C. Reis, V. Borges, B. A. Moser, T. M. Nakamura, and M. G.

Caspari, T., J. M. Murray, and A. M. Carr. 2002. Cdc2-cyclin B kinase activity links Crb2 and

Cejka, P., E. Cannavo, P. Polaczek, T. Masuda-Sasa, S. Pokharel, J. L. Campbell, and S. C.

Chang, S., A. S. Multani, N. G. Cabrera, M. L. Naylor, P. Laud, D. Lombard, S. Pathak, L.

Chavez, A., A. M. Tsou, and F. B. Johnson. 2009. Telomeres do the (un)twist: helicase actions

Cohen, H., and D. A. Sinclair. 2001. Recombination-mediated lengthening of terminal

Cooper, J. P., Y. Watanabe, and P. Nurse. 1998. Fission yeast Taz1 protein is required for meiotic telomere clustering and recombination. *Nature* 392 (6678):828-31. Crabbe, L., A. Jauch, C. M. Naeger, H. Holtgreve-Grez, and J. Karlseder. 2007. Telomere

Crabbe, L., R. E. Verdun, C. I. Haggblom, and J. Karlseder. 2004. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. *Science* 306 (5703):1951-3. Critchlow, S. E., and S. P. Jackson. 1998. DNA end-joining: from yeast to man. *Trends* 

Dewar, J. M., and D. Lydall. 2010. Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping. *EMBO J* 29 (23):4020-34. Diede, S. J., and D. E. Gottschling. 2001. Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere. *Curr Biol* 11 (17):1336-40. Dionne, I., and R. J. Wellinger. 1996. Cell cycle-regulated generation of single-stranded G-

Doe, C. L., J. Dixon, F. Osman, and M. C. Whitby. 2000. Partial suppression of the fission

Faure, V., S. Coulon, J. Hardy, and V. Geli. 2010. Cdc13 and telomerase bind through

pathways regulate 3' overhang generation at *S. cerevisiae* telomeres. *Mol. Cell* 35

Ferreira. 2010. Telomeres avoid end detection by severing the checkpoint signal

Kowalczykowski. 2010. DNA end resection by Dna2-Sgs1-RPA and its stimulation

Guarente, and R. A. DePinho. 2004. Essential role of limiting telomeres in the

telomeric repeats requires the Sgs1 DNA helicase. *Proc Natl Acad Sci U S A* 98

dysfunction as a cause of genomic instability in Werner syndrome. *Proc. Natl. Acad.* 

rich DNA in the absence of telomerase. *Proc. Natl. Acad. Sci. U. S. A.* 93 (24):13902-7.

different mechanisms at the lagging- and leading-strand telomeres. *Mol Cell* 38

phenotype by expression of a bacterial Holliday junction resolvase.

repair. *Annu Rev Genet* 44:393-417.

transduction pathway. *Nature* 467 (7312):228-32.

Rqh1-topoisomerase III. *Genes Dev.* 16 (10):1195-208.

by Top3-Rmi1 and Mre11-Rad50-Xrs2. *Nature* 467 (7311):112-6.

pathogenesis of Werner syndrome. *Nat Genet* 36 (8):877-82.

at chromosome termini. *Biochim Biophys Acta* 1792 (4):329-40.

(1):70-81.

(6):3174-9.

yeast *rqh1*-

(6):842-52.

*Sci. U. S. A.* 104 (7):2205-10.

*Biochem Sci* 23 (10):394-8.

*EMBO J.* 19 (11):2751-62.

Taz1 ortholog) and human RecQ homolog WRN and BLM in telomere maintenance are also suggested (Opresko 2008). TRF2 interacts with WRN and stimulates helicase activity of WRN in vitro (Opresko et al. 2002; Machwe, Xiao, and Orren 2004). Expression of a TRF2 lacking the amino terminal basic domain induces the telomeric circle formations and rapid telomere deletions (Wang, Smogorzewska, and de Lange 2004). These events are dependent on WRN (Li et al. 2008). TRF2 also protects the displacement of Holliday junctions with telomeric arm by WRN in vitro (Nora, Buncher, and Opresko 2010). These facts suggest that the regulation of WRN activity by TRF2 is required to protect telomere.

### **5. Conclusion**

This chapter focused on the roles of proteins involved in the processing of DBS ends at functional and dysfunctional telomere in *S. pombe, S. cerevisiae* and human. We found that MRN, Dna2, and possibly RecQ helicase Rqh1 are involved in the processing at telomere ends in *S. pombe*. Lydall group and other group found that Exo1, RecQ helicase Sgs1, Dna2, and Pif1 are involved in the processing at telomere ends in *S. cerevisiae*. Interestingly, most of these proteins were also involved in the processing of DNA double-strand break ends. These facts raise a new question of how these proteins are regulated at telomere ends. This chapter also focused on the functional interactions between telomere capping proteins and proteins involved in the processing of DBS ends mainly in *S. pombe*. We found that Taz1 and RPA collaborate to inhibit DNA end-processing, possibly by RecQ helicase, to prevent telomere loss. We also found that single-stranded telomere-binding protein Pot1 and RecQ helicase Rqh1 collaborate to inhibit homologous recombination at telomere. Cooper group found that RecQ helicase Rqh1 makes *taz1* disruptant sensitive to cold temperature by creating telomere entanglement. From these analyses, we learned that both double-stranded and single-stranded telomere binding proteins play critical roles to control proteins involved in DNA repair at chromosome ends.

#### **6. Acknowledgment**

I wish to thank all of my collaborators for support on my research. Part of this work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan to Masaru Ueno.

#### **7. References**


Taz1 ortholog) and human RecQ homolog WRN and BLM in telomere maintenance are also suggested (Opresko 2008). TRF2 interacts with WRN and stimulates helicase activity of WRN in vitro (Opresko et al. 2002; Machwe, Xiao, and Orren 2004). Expression of a TRF2 lacking the amino terminal basic domain induces the telomeric circle formations and rapid telomere deletions (Wang, Smogorzewska, and de Lange 2004). These events are dependent on WRN (Li et al. 2008). TRF2 also protects the displacement of Holliday junctions with telomeric arm by WRN in vitro (Nora, Buncher, and Opresko 2010). These facts suggest that

This chapter focused on the roles of proteins involved in the processing of DBS ends at functional and dysfunctional telomere in *S. pombe, S. cerevisiae* and human. We found that MRN, Dna2, and possibly RecQ helicase Rqh1 are involved in the processing at telomere ends in *S. pombe*. Lydall group and other group found that Exo1, RecQ helicase Sgs1, Dna2, and Pif1 are involved in the processing at telomere ends in *S. cerevisiae*. Interestingly, most of these proteins were also involved in the processing of DNA double-strand break ends. These facts raise a new question of how these proteins are regulated at telomere ends. This chapter also focused on the functional interactions between telomere capping proteins and proteins involved in the processing of DBS ends mainly in *S. pombe*. We found that Taz1 and RPA collaborate to inhibit DNA end-processing, possibly by RecQ helicase, to prevent telomere loss. We also found that single-stranded telomere-binding protein Pot1 and RecQ helicase Rqh1 collaborate to inhibit homologous recombination at telomere. Cooper group found that RecQ helicase Rqh1 makes *taz1* disruptant sensitive to cold temperature by creating telomere entanglement. From these analyses, we learned that both double-stranded and single-stranded telomere binding proteins play critical roles to control proteins

I wish to thank all of my collaborators for support on my research. Part of this work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of

Arnoult, N., C. Saintome, I. Ourliac-Garnier, J. F. Riou, and A. Londono-Vallejo. 2009.

Ashton, T. M., and I. D. Hickson. 2010. Yeast as a model system to study RecQ helicase

Barrientos, K. S., M. F. Kendellen, B. D. Freibaum, B. N. Armbruster, K. T. Etheridge, and C.

Baumann, P., and T. R. Cech. 2001. Pot1, the putative telomere end-binding protein in fission

Human POT1 is required for efficient telomere C-rich strand replication in the

M. Counter. 2008. Distinct functions of POT1 at telomeres. *Mol. Cell. Biol.* 28

the regulation of WRN activity by TRF2 is required to protect telomere.

involved in DNA repair at chromosome ends.

Education, Science, Sports and Culture of Japan to Masaru Ueno.

absence of WRN. *Genes Dev.* 23 (24):2915-24.

yeast and humans. *Science* 292 (5519):1171-5.

function. *DNA Repair (Amst)* 9 (3):303-14.

**6. Acknowledgment**

**7. References** 

(17):5251-64.

**5. Conclusion** 


Roles of DNA Repair Proteins in Telomere Maintenance 609

Lee, J. Y., M. Kozak, J. D. Martin, E. Pennock, and F. B. Johnson. 2007. Evidence that a RecQ

Li, B., S. P. Jog, S. Reddy, and L. Comai. 2008. WRN controls formation of extrachromosomal

Lillard-Wetherell, K., A. Machwe, G. T. Langland, K. A. Combs, G. K. Behbehani, S. A.

Liu, N. N., T. X. Han, L. L. Du, and J. Q. Zhou. 2010. A genome-wide screen for

Longhese, M. P., D. Bonetti, N. Manfrini, and M. Clerici. 2010. Mechanisms and regulation

Lydall, D. 2009. Taming the tiger by the tail: modulation of DNA damage responses by

Machwe, A., L. Xiao, and D. K. Orren. 2004. TRF2 recruits the Werner syndrome (WRN) exonuclease for processing of telomeric DNA. *Oncogene* 23 (1):149-56. Makarov, V. L., Y. Hirose, and J. P. Langmore. 1997. Long G tails at both ends of human

Mallory, J. C., V. I. Bashkirov, K. M. Trujillo, J. A. Solinger, M. Dominska, P. Sung, W. D.

Miller, K. M., and J. P. Cooper. 2003. The telomere protein Taz1 is required to prevent and

Miller, K. M., O. Rog, and J. P. Cooper. 2006. Semi-conservative DNA replication through

Mimitou, E. P., and L. S. Symington. 2008. Sae2, Exo1 and Sgs1 collaborate in DNA double-

Mimitou, E. P., and L. S. Symington. 2009. Nucleases and helicases take center stage in

Mimitou, E. P., and L. S. Symington. 2010. Ku prevents Exo1 and Sgs1-dependent resection

Moser, B. A., L. Subramanian, Y. T. Chang, C. Noguchi, E. Noguchi, and T. M. Nakamura.

Multani, A. S., and S. Chang. 2007. WRN at telomeres: implications for aging and cancer. *J.* 

Murayama, Y., Y. Kurokawa, K. Mayanagi, and H. Iwasaki. 2008. Formation and branch

of DNA ends in the absence of a functional MRX complex or Sae2. *EMBO J* 29

2009. Differential arrival of leading and lagging strand DNA polymerases at fission

migration of Holliday junctions mediated by eukaryotic recombinases. *Nature* 451

*Cell Biol* 28 (6):1892-904.

*Mol Genet* 13 (17):1919-32.

of DNA end resection. *EMBO J* 29 (17):2864-74.

repair genomic DNA breaks. *Mol. Cell* 11 (2):303-13.

telomeres requires Taz1. *Nature* 440 (7085):824-8.

strand break processing. *Nature* 455 (7214):770-4.

yeast telomeres. *EMBO. J.* 28 (7):810-20.

*Cell. Sci.* 120 (Pt 5):713-21.

homologous recombination. *Trends Biochem Sci* 34 (5):264-72.

telomeres. *EMBO. J.* 28 (15):2174-2187.

20 (8):963-5.

*Cell* 88 (5):657-66.

(9):1041-64.

(19):3358-69.

(7181):1018-21.

helicase slows senescence by resolving recombining telomeres. *PLoS Biol* 5 (6):e160.

telomeric circles and is required for TRF2DeltaB-mediated telomere shortening. *Mol* 

Schonberg, J. German, J. J. Turchi, D. K. Orren, and J. Groden. 2004. Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2. *Hum* 

Schizosaccharomyces pombe deletion mutants that affect telomere length. *Cell Res*

chromosomes suggest a C strand degradation mechanism for telomere shortening.

Heyer, and T. D. Petes. 2003. Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae. *DNA Repair (Amst)* 2


Firmenich, A. A., M. Elias-Arnanz, and P. Berg. 1995. A novel allele of Saccharomyces

Fishman-Lobell, J., and J. E. Haber. 1992. Removal of nonhomologous DNA ends in double-

Garvik, B., M. Carson, and L. Hartwell. 1995. Single-stranded DNA arising at telomeres in

Gong, Y., and T. de Lange. 2010. A Shld1-controlled POT1a provides support for repression of ATR signaling at telomeres through RPA exclusion. *Mol Cell* 40 (3):377-87. Gravel, S., J. R. Chapman, C. Magill, and S. P. Jackson. 2008. DNA helicases Sgs1 and BLM promote DNA double-strand break resection. *Genes Dev.* 22 (20):2767-72. Gravel, S., M. Larrivee, P. Labrecque, and R. J. Wellinger. 1998. Yeast Ku as a regulator of

Huang, P., F. E. Pryde, D. Lester, R. L. Maddison, R. H. Borts, I. D. Hickson, and E. J. Louis.

Iglesias, N., and J. Lingner. 2009. Related mechanisms for end processing at telomeres and

Ivanov, E. L., and J. E. Haber. 1995. RAD1 and RAD10, but not other excision repair genes,

Ivanov, E. L., N. Sugawara, J. Fishman-Lobell, and J. E. Haber. 1996. Genetic requirements

Johnson, F. B., R. A. Marciniak, M. McVey, S. A. Stewart, W. C. Hahn, and L. Guarente. 2001.

Kibe, T., Y. Ono, K. Sato, and M. Ueno. 2007. Fission yeast Taz1 and RPA are synergistically required to prevent rapid telomere loss. *Mol. Biol. Cell.* 18 (6):2378-87. Kibe, T., K. Tomita, A. Matsuura, D. Izawa, T. Kodaira, T. Ushimaru, M. Uritani, and M.

Kurokawa, Y., Y. Murayama, N. Haruta-Takahashi, I. Urabe, and H. Iwasaki. 2008.

Larrivee, M., C. LeBel, and R. J. Wellinger. 2004. The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex. *Genes Dev.* 18 (12):1391-6. Laud, P. R., A. S. Multani, S. M. Bailey, L. Wu, J. Ma, C. Kingsley, M. Lebel, S. Pathak, R. A.

engagement of the ALT pathway. *Genes Dev.* 19 (21):2560-70.

maintenance in cells lacking telomerase. *EMBO J* 20 (4):905-13.

2001. SGS1 is required for telomere elongation in the absence of telomerase. *Curr* 

are required for double-strand break-induced recombination in Saccharomyces

for the single-strand annealing pathway of double-strand break repair in

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere

Ueno. 2003. Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer. *Nucleic Acids Res.* 31 (17):5054-63. Kobayashi, Y., K. Sato, T. Kibe, H. Seimiya, A. Nakamura, M. Yukawa, E. Tsuchiya, and M.

Ueno. 2010. Expression of mutant RPA in human cancer cells causes telomere

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two

DePinho, and S. Chang. 2005. Elevated telomere-telomere recombination in WRNdeficient, telomere dysfunctional cells promotes escape from senescence and

chromosomal DNA end structure. *Science* 280 (5364):741-4.

DNA double-strand breaks. *Mol Cell* 35 (2):137-8.

Saccharomyces cerevisiae. *Genetics* 142 (3):693-704.

shortening. *Biosci Biotechnol Biochem* 74 (2):382-5.

mediators. *PLoS Biol* 6 (4):e88.

cerevisiae. *Mol Cell Biol* 15 (4):2245-51.

RAD52. *Mol Cell Biol* 15 (3):1620-31.

*Science* 258 (5081):480-4.

*Biol.* 15 (11):6128-38.

*Biol* 11 (2):125-9.

cerevisiae RFA1 that is deficient in recombination and repair and suppressible by

strand break recombination: the role of the yeast ultraviolet repair gene RAD1.

cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. *Mol. Cell.* 


Roles of DNA Repair Proteins in Telomere Maintenance 611

Parker, A. E., R. K. Clyne, A. M. Carr, and T. J. Kelly. 1997. The *Schizosaccharomyces pombe*

Polotnianka, R. M., J. Li, and A. J. Lustig. 1998. The yeast Ku heterodimer is essential for

Rog, O., K. M. Miller, M. G. Ferreira, and J. P. Cooper. 2009. Sumoylation of RecQ helicase

Rossi, M. L., A. K. Ghosh, and V. A. Bohr. 2010. Roles of Werner syndrome protein in

Schramke, V., P. Luciano, V. Brevet, S. Guillot, Y. Corda, M. P. Longhese, E. Gilson, and V.

Shim, E. Y., W. H. Chung, M. L. Nicolette, Y. Zhang, M. Davis, Z. Zhu, T. T. Paull, G. Ira,

Smith, J., H. Zou, and R. Rothstein. 2000. Characterization of genetic interactions with *RFA1*:

Sowd, G., M. Lei, and P. L. Opresko. 2008. Mechanism and substrate specificity of telomeric

Stewart, E., C. R. Chapman, F. Al-Khodairy, A. M. Carr, and T. Enoch. 1997. *rqh1+*, a fission

Sugawara, N., G. Ira, and J. E. Haber. 2000. DNA length dependence of the single-strand

Takahashi, K., R. Imano, T. Kibe, H. Seimiya, Y. Muramatsu, N. Kawabata, G. Tanaka, Y.

Tomita, K., T. Kibe, H. Y. Kang, Y. S. Seo, M. Uritani, T. Ushimaru, and M. Ueno. 2004.

Tomita, K., A. Matsuura, T. Caspari, A. M. Carr, Y. Akamatsu, H. Iwasaki, K. Mizuno, K.

efficient chromosome segregation. *Mol Cell Biol* 31 (3):495-506.

controls the fate of dysfunctional telomeres. *Mol. Cell* 33 (5):559-69.

protection of genome integrity. *DNA Repair (Amst)* 9 (3):331-44.

chromosome ends. *Nat. Genet.* 36 (1):46-54.

reversible S phase arrest. *EMBO J.* 16 (10):2682-92.

strand break repair. *Mol Cell Biol* 20 (14):5300-9.

overhang. *Mol Cell Biol* 24 (21):9557-67.

(5):2381-90.

*Biol* 8 (14):831-4.

(13):4242-56.

1005.

*rad11*+ gene encodes the large subunit of replication protein A. *Mol. Cell. Biol.* 17

protection of the telomere against nucleolytic and recombinational activities. *Curr* 

Geli. 2004. RPA regulates telomerase action by providing Est1p access to

and S. E. Lee. 2010. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. *EMBO J* 29 (19):3370-80. Smith, J., and R. Rothstein. 1995. A mutation in the gene encoding the *Saccharomyces* 

*cerevisiae* single-stranded DNA-binding protein Rfa1 stimulates a RAD52 independent pathway for direct-repeat recombination. *Mol. Cell. Biol.* 15 (3):1632-41.

the role of RPA in DNA replication and telomere maintenance. *Biochimie* 82 (1):71-8.

protein POT1 stimulation of the Werner syndrome helicase. *Nucleic Acids Res* 36

yeast gene related to the Bloom's and Werner's syndrome genes, is required for

annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-

Matsumoto, T. Hiromoto, Y. Koizumi, N. Nakazawa, M. Yanagida, M. Yukawa, E. Tsuchiya, and M. Ueno. 2011. Fission yeast Pot1 and RecQ helicase are required for

Fission yeast Dna2 is required for generation of the telomeric single-strand

Ohta, M. Uritani, T. Ushimaru, K. Yoshinaga, and M. Ueno. 2003. Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres. *Mol Cell Biol* 23 (15):5186-97. Umezu, K., N. Sugawara, C. Chen, J. E. Haber, and R. D. Kolodner. 1998. Genetic analysis of

yeast RPA1 reveals its multiple functions in DNA metabolism. *Genetics* 148 (3):989-


Murray, J. M., H. D. Lindsay, C. A. Munday, and A. M. Carr. 1997. Role of

Nakamura, T. M., J. P. Cooper, and T. R. Cech. 1998. Two modes of survival of fission yeast

Ngo, H. P., and D. Lydall. 2010. Survival and growth of yeast without telomere capping by Cdc13 in the absence of Sgs1, Exo1, and Rad9. *PLoS Genet* 6 (8):e1001072. Nimonkar, A. V., J. Genschel, E. Kinoshita, P. Polaczek, J. L. Campbell, C. Wyman, P.

Niu, H., W. H. Chung, Z. Zhu, Y. Kwon, W. Zhao, P. Chi, R. Prakash, C. Seong, D. Liu, L.

Ono, Y., K. Tomita, A. Matsuura, T. Nakagawa, H. Masukata, M. Uritani, T. Ushimaru, and

Opresko, P. L. 2008. Telomere ResQue and preservation--roles for the Werner syndrome

Opresko, P. L., P. A. Mason, E. R. Podell, M. Lei, I. D. Hickson, T. R. Cech, and V. A. Bohr.

Opresko, P. L., M. Otterlei, J. Graakjaer, P. Bruheim, L. Dawut, S. Kolvraa, A. May, M. M.

Opresko, P. L., G. Sowd, and H. Wang. 2009. The Werner syndrome helicase/exonuclease

Opresko, P. L., C. von Kobbe, J. P. Laine, J. Harrigan, I. D. Hickson, and V. A. Bohr. 2002.

Ouellette, M. M., L. D. McDaniel, W. E. Wright, J. W. Shay, and R. A. Schultz. 2000. The

Palm, W., and T. de Lange. 2008. How shelterin protects mammalian telomeres. *Annu. Rev.* 

Paques, F., and J. E. Haber. 1997. Two pathways for removal of nonhomologous DNA ends

chromosome instability syndromes. *Hum Mol Genet* 9 (3):403-11.

protein and other RecQ helicases. *Mech. Ageing Dev.* 129 (1-2):79-90.

repair and telomere length regulation. *Nucleic Acids Res.* 31 (24):7141-9. Onoda, F., M. Seki, A. Miyajima, and T. Enomoto. 2001. Involvement of SGS1 in DNA

UV damage tolerance. *Mol. Cell. Biol.* 17 (12):6868-75.

syndrome helicase. *Nucleic Acids Res* 38 (12):3984-98.

Saccharomyces cerevisiae. *Mol Gen Genet* 264 (5):702-8.

substrates. *J. Biol. Chem.* 280 (37):32069-80.

syndrome helicases. *J. Biol. Chem.* 277 (43):41110-9.

*Mol Cell* 14 (6):763-74.

(3):e4825.

*Genet.* 42:301-34.

(11):6765-71.

without telomerase. *Science* 282 (5388):493-6.

break repair. *Genes Dev* 25 (4):350-62.

*Schizosaccharomyces pombe* RecQ homolog, recombination, and checkpoint genes in

Modrich, and S. C. Kowalczykowski. 2011. BLM-DNA2-RPA-MRN and EXO1- BLM-RPA-MRN constitute two DNA end resection machineries for human DNA

Lu, G. Ira, and P. Sung. 2010. Mechanism of the ATP-dependent DNA endresection machinery from Saccharomyces cerevisiae. *Nature* 467 (7311):108-11. Nora, G. J., N. A. Buncher, and P. L. Opresko. 2010. Telomeric protein TRF2 protects

Holliday junctions with telomeric arms from displacement by the Werner

M. Ueno. 2003. A novel allele of fission yeast *rad11* that causes defects in DNA

damage-induced heteroallelic recombination that requires RAD52 in

2005. POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA

Seidman, and V. A. Bohr. 2004. The Werner syndrome helicase and exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 and TRF2.

processes mobile D-loops through branch migration and degradation. *PLoS One* 4

Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom

establishment of telomerase-immortalized cell lines representing human

during double-strand break repair in Saccharomyces cerevisiae. *Mol Cell Biol* 17


**Part 6** 

**Measuring DNA Repair Capacity** 


**Part 6** 

**Measuring DNA Repair Capacity** 

612 DNA Repair

Verdun, R. E., and J. Karlseder. 2006. The DNA damage machinery and homologous

Wang, R. C., A. Smogorzewska, and T. de Lange. 2004. Homologous recombination generates T-loop-sized deletions at human telomeres. *Cell* 119 (3):355-68. Wang, X., and P. Baumann. 2008. Chromosome fusions following telomere loss are mediated

Watt, P. M., I. D. Hickson, R. H. Borts, and E. J. Louis. 1996. SGS1, a homologue of the

Wellinger, R. J., K. Ethier, P. Labrecque, and V. A. Zakian. 1996. Evidence for a new step in

Wellinger, R. J., A. J. Wolf, and V. A. Zakian. 1993. *Saccharomyces* telomeres acquire single-

Wu, L., and I. D. Hickson. 2003. The Bloom's syndrome helicase suppresses crossing over

Wyllie, F. S., C. J. Jones, J. W. Skinner, M. F. Haughton, C. Wallis, D. Wynford-Thomas, R. G.

Zaug, A. J., E. R. Podell, and T. R. Cech. 2005. Human POT1 disrupts telomeric G-

Zhu, Z., W. H. Chung, E. Y. Shim, S. E. Lee, and G. Ira. 2008. Sgs1 helicase and two

Zou, L., and S. J. Elledge. 2003. Sensing DNA damage through ATRIP recognition of RPA-

Faragher, and D. Kipling. 2000. Telomerase prevents the accelerated cell ageing of

quadruplexes allowing telomerase extension in vitro. *Proc Natl Acad Sci U S A* 102

nucleases Dna2 and Exo1 resect DNA double-strand break ends. *Cell* 134 (6):981-94.

by single-strand annealing. *Mol. Cell* 31 (4):463-73.

strand TG1-3 tails late in S phase. *Cell* 72 (1):51-60.

Werner syndrome fibroblasts. *Nat Genet* 24 (1):16-7.

ssDNA complexes. *Science* 300 (5625):1542-8.

telomere maintenance. *Cell* 85 (3):423-33.

stability in Saccharomyces cerevisiae. *Genetics* 144 (3):935-45.

during homologous recombination. *Nature* 426 (6968):870-4.

(4):709-20.

(31):10864-9.

recombination pathway act consecutively to protect human telomeres. *Cell* 127

Bloom's and Werner's syndrome genes, is required for maintenance of genome

**31** 

*1Spain 2Norway* 

*1University of Navarra 2University of Oslo*

**DNA Repair Measured by the Comet Assay** 

Amaya Azqueta1, Sergey Shaposhnikov2 and Andrew R. Collins2

The stability of the genome is of crucial importance, and yet the DNA molecule is prone to spontaneous loss of bases, and damage from exogenous and endogenous sources – with potentially mutagenic consequences. Damage can take the form of small alterations to bases (alkylation or oxidation); breaks in the sugar-phosphate backbone involving one or both strands (single or double strand breaks – SSBs or DSBs); bulky adducts combined with bases; and covalent bonds between adjacent bases (intra-strand cross-links), across the double helix (inter-strand cross-links), or between DNA and protein. These lesions can

Cells possess repair enzymes that correct almost all the damage before it can result in permanent change to the genome. Different pathways deal with the various kinds of damage. Repair of SSBs is in most cells a rapid process, consisting of little more than ligation. DSBs are more complicated (and potentially more serious) since the continuity of the double helix is disrupted. Homologous recombination ensures restoration of the correct DNA sequence by using the DNA of the sister chromatid or homologous chromosome as a template, while non-homologous end-rejoining is less precise and can entail loss of sequence. Base excision repair (BER) is concerned with small base alterations and starts with removal of the damaged base by a more or less specific glycosylase, leaving a base-less sugar or AP-site (apurinic/apyrimidinic site). An AP endonuclease or lyase cleaves the DNA at this site, and – after trimming of the broken ends of DNA – the one-nucleotide gap is filled by DNA polymerase β. Ligation is the final stage. Nucleotide excision repair (NER) is a more complex affair, involving recognition of a bulky adduct or helix distortion (such as is caused by the dimerisation of adjacent pyrimidines by UV(C) radiation), endonucleolytic incision on each side of the lesion, and removal of an oligonucleotide containing the damage. This is then filled in by DNA polymerase δ, κ or ε and the new patch of nucleotides is ligated into the DNA, completing the repair. NER enzymes are also involved in repair of inter-strand cross-links, removing the linking molecule from one strand, leaving it attached to the other strand as a mono-adduct to be removed in a second NER reaction

Individual DNA repair capacity is regarded as a biomarker of susceptibility to mutation and cancer. A person with high repair rate is assumed to be at lower risk than one with low repair rate. DNA repair is partially determined genetically, and polymorphisms in repair

disrupt replication, or cause incorporation of the wrong base.

(according to the simplest, and possibly simplistic, model).

**1. Introduction** 
