**6. TopBP1 and activation of ATR pathway**

The major regulators of DNA damage response are the phosphoinositide 3-kinase (PI3K) related proteins kinases (PIKKs), including ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) (Cimprich & Cortez, 2008; Lopez-Contreras & Fernandez-Capetillo, 2010; Takeishi et al., 2010). Other members of this family comprise mTOR (mammalian target of rapamycin), which coordinates protein synthesis and cell growth, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), which promotes DNA double strand break repair by nonhomologous end-joining and SMG1, which regulates nonsense-mediated mRNA decay (Cimprich & Cortez, 2008; Mordes et al., 2008). PIKKs are large proteins (2549 – 4128 amino acids) with common domain architecture. All of them contain a large region of repeated HEAT (Huntington, elongation factor 3, PR65/A, TOR) domains in the N terminus, highly conserved C-terminal kinase domain flanked by FAT (FRAP, ATM, TRAP /FKBP-rapamycin associated protein, ATM, *trp* RNA binding attenuation protein) and FATC (FAT C terminus) and PIKK regulatory domain (PRD) between the kinase and FATC domains (Cimprich & Cortez, 2008; Lopez-Contreras & Fernandez-Capetillo, 2010; Mordes et al., 2008). PRD, poorly conserved between family members but highly conserved within orthologous present in different organisms, is not essential for basal kinase activity but plays a regulatory role in at least ATM, ATR and mTOR (Cimprich & Cortez, 2008). PRD of ATM and mTOR is targeted for post-translational modifications that regulate their activity (Cimprich & Cortez, 2008; Mordes et al., 2008). The N-terminal regions of the kinases mediate interaction with the protein cofactors (Cimprich & Cortez, 2008). ATM and ATR are proteins of about 300 kDa, with a conserved C-terminal catalytic domain that preferably phosphorylates serine or threonine residues followed by a glutamine, i.e. SQ or TQ motif (Choi et al., 2009; Smits et al., 2010).

The initial step in ATR activation is recognition of DNA structures that are induced by the damaging agents (Smits et al., 2010). As mentioned, ATR responds to a wide variety of DNA damage that results in the generation of single-stranded DNA (ssDNA) (Takeishi et al., 2010). In eukaryotes, DNA damage-induced ssDNA is first detected by ssDNA binding protein complex RPA (Fig. 3) (Smits et al., 2010). RPA is a heterotrimeric protein complex composed of three subunits with a size of 70, 30 and 14 kDa, which are known as RPA70, RPA32 and RPA14 or alternatively RPA1, RPA2 and RPA3, respectively (Binz et al., 2004; Broderick et al., 2010; Fanning et al., 2006). RPA is identified to be a crucial component in DNA replication, DNA recombination and DNA repair (Ball et al., 2007; Broderick et al., 2010; Cimprich & Cortez, 2008). After binding to ssDNA either during DNA replication or in response to DNA damage, RPA is phosphorylated and this is thought to be an important event in DNA damage response (Binz et al., 2004; Broderick et al., 2010). Recent observations have shown the involvement of ATR in the RPA2 phosphorylation in response to stalled replication fork in S phase generated by genotoxic agents such as UV (Broderick et al., 2010; Olson et al., 2006).

RPA-coated ssDNA is necessary for ATR activation, but it is not sufficient, as at least several additional factors are also required. This kinase forms a stable complex with ATRIP (ATRinteracting protein) which regulates the localization of ATR to sites of replication stress and

TopBP1 in DNA Damage Response 293

region have no effect on the basal activity of ATR, although they prevent ATR activation by TopBP1 protein and cause checkpoint defects and mimic a complete deletion of ATR in human somatic cells (Cimprich & Cortez, 2008; Mordes et al., 2008). Thus, efficient activation of ATR by TopBP1 protein may be required to achieve sufficient signal amplification for the proper

Fig. 3. Role of TopBP1 in activation of ATR pathway in response to replication stress and

UV-induced DNA damage

execution of cellular response to DNA damage (Sokka et al., 2010).

DNA damage. Apart from ATRIP, activation of ATR requires the activator protein TopBP1 which plays dual role in the initiation of DNA replication and DNA damage response (Mordes & Cortez, 2008). ATRIP was identified as a 85 kDa an ATR binding partner that interacts directly with RPA to dock the ATR-ATRIP complex onto ssDNA (Ball et al., 2007; Choi et al., 2010; Kim et al., 2005; Warmerdam & Kanaar, 2010; Yan & Michael, 2009a,b). Independently, the Rad17-RFC complex is loaded onto these sites of damage in RPAdependent manner (Burrows & Elledge, 2008; Lee & Dunphy, 2010). The Rad17-RFC complex consists of the Rad17 subunit and four additional subunits of replication factor C named from RFC2 to RFC5. During normal replication the RFC complex, containing RFC1 instead of Rad17, plays a role in the loading of PCNA onto DNA. PCNA is a processivity factor for DNA polymerases. Both the Rad17 and RFC complexes require RPA for their loading onto DNA (Majka et al., 2006; Medhurst et al., 2008; Warmerdam & Kanaar, 2010). However, Rad17-RFC requires 5' dsDNA-ssDNA junctions, rather than the 3' ended junctions preferred by PCNA. These types of structures are specifically created by the resection of DSBs, stalled replication forks and UV-induced ssDNA gaps. The Rad17-RFC protein complex facilitates the loading of the Rad9-Rad1-Hus1 (9-1-1) sliding clamp onto the DNA (Choi et al., 2010; Lopez-Contreras & Fernandez-Capetillo, 2010; Van et al., 2010; Warmerdam & Kanaar, 2010; Yan & Michael, 2009a). The necessity of the 9-1-1 complex in the ATR branch was explained by showing that Rad9 recruits the ATR-activator TopBP1 protein near sites of DNA damage, which was consistent with earlier reports showing interaction between Rad9 and TopBP1 protein (Greer et al., 2003; Makiniemi et al., 2001; Smits et al., 2010). The amino-terminal region of TopBP1 protein comprising BRCT1 and BRCT2 binds the C terminus of Rad9. More precisely, the interaction between Rad9 and TopBP1 depends on Ser373 phosphorylation in the C-terminal tail of Rad9 (Delacroix et al., 2007; Kumagai et al., 2006; Lee et al., 2007; Rappas et al., 2011; Smits et al., 2010; Takeishi et al., 2010). Then, TopBP1 protein binds ATR through its ATR activation domain (AAD), located between the sixth and seventh BRCT repeats, in an ATRIP-dependent manner and this interaction is required for ATR stimulation (Kumagai et al, 2006; Mordes et al., 2008; Smits et al., 2010; Takeishi et al., 2010). ATRIP contains a conserved TopBP1 interacting region, required for the association of TopBP1 and ATR and the subsequent TopBP1 mediated triggering of ATR activity (Mordes et al., 2008; Smits et al., 2010).

ATR-mediated activation of Chk1 in response to genotoxic stress requires another protein that binds independently of ATR or Rad17/9-1-1 named Claspin (Kumagai et al., 2004; Liu et al., 2006b; Scorah & McGowan, 2009; Smits et al., 2010). Claspin is proposed to function as adaptor molecule bringing ATR and Chk1 together (Kumagai & Dunphy, 2000; Smits et al., 2010). The Claspin-Chk1 interaction depends on ATR-mediated phosphorylation of Claspin and is required for Chk1 phosphorylation by ATR. Subsequent studies identified repeated phosphopeptide motifs in Claspin, which are required for association with phosphate binding sites in the N-terminal kinase domain of Chk1, resulting in full activation of Chk1 (Smits et al., 2010). In response to DNA damage or replication stress activated ATR and its effectors such as Chk1 ultimately slow origin firing and induce cell cycle arrest, as well as stabilize and restart stalled replication forks (Cimprich & Cortez, 2008).

The mechanism by which TopBP1 binding activates ATR is poorly defined. The primary binding site for the activation domain of TopBP1 on the ATR complex is within ATRIP and mutations in this region of ATRIP block activation (Cimprich & Cortez, 2008; Mordes et al., 2008). In addition, activation involves amino acids that are located between the ATR kinase domain and the FATC domain, of PIKK regulatory domain - PRD of ATR. Mutations in this

DNA damage. Apart from ATRIP, activation of ATR requires the activator protein TopBP1 which plays dual role in the initiation of DNA replication and DNA damage response (Mordes & Cortez, 2008). ATRIP was identified as a 85 kDa an ATR binding partner that interacts directly with RPA to dock the ATR-ATRIP complex onto ssDNA (Ball et al., 2007; Choi et al., 2010; Kim et al., 2005; Warmerdam & Kanaar, 2010; Yan & Michael, 2009a,b). Independently, the Rad17-RFC complex is loaded onto these sites of damage in RPAdependent manner (Burrows & Elledge, 2008; Lee & Dunphy, 2010). The Rad17-RFC complex consists of the Rad17 subunit and four additional subunits of replication factor C named from RFC2 to RFC5. During normal replication the RFC complex, containing RFC1 instead of Rad17, plays a role in the loading of PCNA onto DNA. PCNA is a processivity factor for DNA polymerases. Both the Rad17 and RFC complexes require RPA for their loading onto DNA (Majka et al., 2006; Medhurst et al., 2008; Warmerdam & Kanaar, 2010). However, Rad17-RFC requires 5' dsDNA-ssDNA junctions, rather than the 3' ended junctions preferred by PCNA. These types of structures are specifically created by the resection of DSBs, stalled replication forks and UV-induced ssDNA gaps. The Rad17-RFC protein complex facilitates the loading of the Rad9-Rad1-Hus1 (9-1-1) sliding clamp onto the DNA (Choi et al., 2010; Lopez-Contreras & Fernandez-Capetillo, 2010; Van et al., 2010; Warmerdam & Kanaar, 2010; Yan & Michael, 2009a). The necessity of the 9-1-1 complex in the ATR branch was explained by showing that Rad9 recruits the ATR-activator TopBP1 protein near sites of DNA damage, which was consistent with earlier reports showing interaction between Rad9 and TopBP1 protein (Greer et al., 2003; Makiniemi et al., 2001; Smits et al., 2010). The amino-terminal region of TopBP1 protein comprising BRCT1 and BRCT2 binds the C terminus of Rad9. More precisely, the interaction between Rad9 and TopBP1 depends on Ser373 phosphorylation in the C-terminal tail of Rad9 (Delacroix et al., 2007; Kumagai et al., 2006; Lee et al., 2007; Rappas et al., 2011; Smits et al., 2010; Takeishi et al., 2010). Then, TopBP1 protein binds ATR through its ATR activation domain (AAD), located between the sixth and seventh BRCT repeats, in an ATRIP-dependent manner and this interaction is required for ATR stimulation (Kumagai et al, 2006; Mordes et al., 2008; Smits et al., 2010; Takeishi et al., 2010). ATRIP contains a conserved TopBP1 interacting region, required for the association of TopBP1 and ATR and the subsequent TopBP1-

mediated triggering of ATR activity (Mordes et al., 2008; Smits et al., 2010).

stabilize and restart stalled replication forks (Cimprich & Cortez, 2008).

ATR-mediated activation of Chk1 in response to genotoxic stress requires another protein that binds independently of ATR or Rad17/9-1-1 named Claspin (Kumagai et al., 2004; Liu et al., 2006b; Scorah & McGowan, 2009; Smits et al., 2010). Claspin is proposed to function as adaptor molecule bringing ATR and Chk1 together (Kumagai & Dunphy, 2000; Smits et al., 2010). The Claspin-Chk1 interaction depends on ATR-mediated phosphorylation of Claspin and is required for Chk1 phosphorylation by ATR. Subsequent studies identified repeated phosphopeptide motifs in Claspin, which are required for association with phosphate binding sites in the N-terminal kinase domain of Chk1, resulting in full activation of Chk1 (Smits et al., 2010). In response to DNA damage or replication stress activated ATR and its effectors such as Chk1 ultimately slow origin firing and induce cell cycle arrest, as well as

The mechanism by which TopBP1 binding activates ATR is poorly defined. The primary binding site for the activation domain of TopBP1 on the ATR complex is within ATRIP and mutations in this region of ATRIP block activation (Cimprich & Cortez, 2008; Mordes et al., 2008). In addition, activation involves amino acids that are located between the ATR kinase domain and the FATC domain, of PIKK regulatory domain - PRD of ATR. Mutations in this region have no effect on the basal activity of ATR, although they prevent ATR activation by TopBP1 protein and cause checkpoint defects and mimic a complete deletion of ATR in human somatic cells (Cimprich & Cortez, 2008; Mordes et al., 2008). Thus, efficient activation of ATR by TopBP1 protein may be required to achieve sufficient signal amplification for the proper execution of cellular response to DNA damage (Sokka et al., 2010).

Fig. 3. Role of TopBP1 in activation of ATR pathway in response to replication stress and UV-induced DNA damage

TopBP1 in DNA Damage Response 295

Fig. 4. Role of TopBP1 in ATR activation in response to DNA double strand breaks

suggest that the BRCT-dependent association of these proteins is critical for normal checkpoint response to DSB (Morishima et al., 2007; Yoo et al., 2009). The MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs (Morishima et al., 2007; Yoo et al., 2009). In *Xenopus* egg extracts, ATM associates with TopBP1 protein and phosphorylates it on Ser1131. This

#### **7. Role of TopBP1 in DSB repair**

TopBP1 protein also plays a direct and essential role in the pathway that connects ATM to ATR, specifically in response to the occurrence of DSBs in a genome (Yoo et al., 2007). DNA double strand breaks are among the most deleterious DNA lesions that threaten genomic integrity. DSBs are generated not only by exogenous DNA-damaging agents, but also by normal cellular processes, such as V(D)J recombination, meiosis and DNA replication. Furthermore, increased amounts of DSBs are induced by oncogenic stresses during the early stage at tumorigenesis (O'Driscoll & Jeggo, 2005; Shiotani & Zou, 2009; Williams et al., 2007). Two major forms of DSB repair are found within eukaryotic cells: nonhomologous endjoining (NHEJ) and homologous recombination (HR). NHEJ requires several complementary bases for repair and is the predominant form of DSB repair in G0/G1 cells. During NHEJ DNA ends are minimally processed to reveal short stretches of complementarity on either side of the break. NHEJ pathway is inherently mutagenic. In contrast, HR pathway predominates during S and G2 phases and repairs DNA with high fidelity by employing homologous chromosomal or sister chromatid DNA as a template to synthesize new error-free DNA (Williams et al., 2007). The main PIKK that responds to DSBs is ATM, the protein that is defective in the hereditary disorder ataxia telangiectasia (O'Driscoll & Jeggo, 2005). DSBs are recognized by the MRE11-RAD50-NBS1 complex, which promotes the activation of ATM and the preparation of DNA ends for homologous recombination (Fig. 4) (Ciccia & Elledge, 2010; O'Driscoll & Jeggo, 2005; Williams et al., 2007). RAD50 contains ATPase domains that interact with MRE11 (meiotic recombination 11) and associates with the DNA ends. MRE11 has endonuclease and exonuclease activities important for the initial step of DNA end resection that is essential for homologous recombination (Ciccia & Elledge, 2010; Williams et al., 2007). The third subunit of the MRN complex, NBS1, interacts with MRE11 and contains additional protein-protein interaction domains important for MRN function in DNA damage response. NBS1 associates with ATM *via* its C-terminal region, which promotes the recruitment of ATM to DSBs, where ATM is activated by the MRN complex (Ciccia & Elledge, 2010; Jazayeri et al., 2008; Kanaar & Wyman, 2008; Rupnik et al., 2010). Mutations in the human *NBS1* gene result in Nijmegen breakage syndrome (NBS), a rare disorder with abnormal responses to ionizing radiation that resemble those in patients with ataxia telangiectasia (Horton et al., 2011). DNA end resection is regulated by ATM through CtIP (C-terminal binding protein/CtBP interacting protein), which interacts with BRCA1 and MRN (Ciccia & Elledge, 2010). In addition, Exo1 (exonuclease 1), which is involved in the processive stage of DSB resection together with BLM following the initial resection carried out by CtIP, is also stimulated by ATM phosphorylation (Bolderson et al., 2010; Ciccia & Elledge, 2010; Shiotani et al., 2009; Smits et al., 2010). DSB resection and formation of 3' ssDNA ends leads to RPA accumulation. RPA-ssDNA complexes play a critical role in activation of ATR pathway, as described in detail above.

TopBP1 protein appeared to be involved in ATR-dependent DSB repair. In human cells, DSB induces formation of distinct TopBP1 foci that colocalize with BRCA1, PCNA, NBS1, 53BP1 and γH2AX (Germann et al., 2011). *In vitro* studies showed that in nuclear foci, TopBP1 protein physically associates with NBS1. Several of TopBP1 foci increased and colocalized with NBS1 after ionizing radiation, whereas these nuclear foci were not observed in Nijmegen breakage syndrome cells. The association between TopBP1 and NBS1 involves the first pair of BRCT repeats in TopBP1. In addition the two tandem BRCT repeats of NBS1 are required for this binding. Functional studies with mutated forms of TopBP1 and NBS1

TopBP1 protein also plays a direct and essential role in the pathway that connects ATM to ATR, specifically in response to the occurrence of DSBs in a genome (Yoo et al., 2007). DNA double strand breaks are among the most deleterious DNA lesions that threaten genomic integrity. DSBs are generated not only by exogenous DNA-damaging agents, but also by normal cellular processes, such as V(D)J recombination, meiosis and DNA replication. Furthermore, increased amounts of DSBs are induced by oncogenic stresses during the early stage at tumorigenesis (O'Driscoll & Jeggo, 2005; Shiotani & Zou, 2009; Williams et al., 2007). Two major forms of DSB repair are found within eukaryotic cells: nonhomologous endjoining (NHEJ) and homologous recombination (HR). NHEJ requires several complementary bases for repair and is the predominant form of DSB repair in G0/G1 cells. During NHEJ DNA ends are minimally processed to reveal short stretches of complementarity on either side of the break. NHEJ pathway is inherently mutagenic. In contrast, HR pathway predominates during S and G2 phases and repairs DNA with high fidelity by employing homologous chromosomal or sister chromatid DNA as a template to synthesize new error-free DNA (Williams et al., 2007). The main PIKK that responds to DSBs is ATM, the protein that is defective in the hereditary disorder ataxia telangiectasia (O'Driscoll & Jeggo, 2005). DSBs are recognized by the MRE11-RAD50-NBS1 complex, which promotes the activation of ATM and the preparation of DNA ends for homologous recombination (Fig. 4) (Ciccia & Elledge, 2010; O'Driscoll & Jeggo, 2005; Williams et al., 2007). RAD50 contains ATPase domains that interact with MRE11 (meiotic recombination 11) and associates with the DNA ends. MRE11 has endonuclease and exonuclease activities important for the initial step of DNA end resection that is essential for homologous recombination (Ciccia & Elledge, 2010; Williams et al., 2007). The third subunit of the MRN complex, NBS1, interacts with MRE11 and contains additional protein-protein interaction domains important for MRN function in DNA damage response. NBS1 associates with ATM *via* its C-terminal region, which promotes the recruitment of ATM to DSBs, where ATM is activated by the MRN complex (Ciccia & Elledge, 2010; Jazayeri et al., 2008; Kanaar & Wyman, 2008; Rupnik et al., 2010). Mutations in the human *NBS1* gene result in Nijmegen breakage syndrome (NBS), a rare disorder with abnormal responses to ionizing radiation that resemble those in patients with ataxia telangiectasia (Horton et al., 2011). DNA end resection is regulated by ATM through CtIP (C-terminal binding protein/CtBP interacting protein), which interacts with BRCA1 and MRN (Ciccia & Elledge, 2010). In addition, Exo1 (exonuclease 1), which is involved in the processive stage of DSB resection together with BLM following the initial resection carried out by CtIP, is also stimulated by ATM phosphorylation (Bolderson et al., 2010; Ciccia & Elledge, 2010; Shiotani et al., 2009; Smits et al., 2010). DSB resection and formation of 3' ssDNA ends leads to RPA accumulation. RPA-ssDNA complexes

play a critical role in activation of ATR pathway, as described in detail above.

TopBP1 protein appeared to be involved in ATR-dependent DSB repair. In human cells, DSB induces formation of distinct TopBP1 foci that colocalize with BRCA1, PCNA, NBS1, 53BP1 and γH2AX (Germann et al., 2011). *In vitro* studies showed that in nuclear foci, TopBP1 protein physically associates with NBS1. Several of TopBP1 foci increased and colocalized with NBS1 after ionizing radiation, whereas these nuclear foci were not observed in Nijmegen breakage syndrome cells. The association between TopBP1 and NBS1 involves the first pair of BRCT repeats in TopBP1. In addition the two tandem BRCT repeats of NBS1 are required for this binding. Functional studies with mutated forms of TopBP1 and NBS1

**7. Role of TopBP1 in DSB repair** 

Fig. 4. Role of TopBP1 in ATR activation in response to DNA double strand breaks

suggest that the BRCT-dependent association of these proteins is critical for normal checkpoint response to DSB (Morishima et al., 2007; Yoo et al., 2009). The MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs (Morishima et al., 2007; Yoo et al., 2009). In *Xenopus* egg extracts, ATM associates with TopBP1 protein and phosphorylates it on Ser1131. This

TopBP1 in DNA Damage Response 297

Boner W., Taylor E.R., Tsirimonaki E., Yamane K., Campo M.S. & Morgan I.M. (2002). A

Broderic S., Rehmet K., Concannon C. & Nasheuer H.P. (2010), In: *Genome stability and* 

Burrows A.E. & Elledge S.J. (2008). How ATR turns on: TopBP1 goes on ATRIP with ATR. *Genes and Development*, Vol. 22, No. 11, (June 2008), pp. 1416-1421, ISSN 0890-9369 Chen H.Z., Tsai S.Y. & Leone G. (2009). Emerging roles of E2Fs in cancer: an exit from cell

Choi J.H., Lindsey-Boltz L.A., Kemp M., Mason A.C., Wold M.S. & Sancar A. (2010).

Choi J.H., Sancar A., Lindsey-Boltz L.A. (2009). The human ATR-mediated DNA damage

Choudhary C., Kumar C., Gnad F., Nielsen M.L., Rehman M., Walther T.C., Olsen J.V. &

Ciccia A. & Elledge S.J. (2010). The DNA damage response: making it safe to play with knives. *Molecular Cell*, Vol. 40, No. 2, (October 2010), pp. 179-204, ISSN: 1097-2765 Cimprich K.A. & Cortez D. (2008). ATR: an essential regulator of genome integrity. *Nature* 

Corre I., Niaudet C. & Paris F. (2010). Plasma membrane signaling induced by ionizing

Cortez D., Gutunku S., Qin J. & Elledge S.J. (2001). ATR and ATRIP: partners in checkpoint signaling. *Science*, Vol. 294, No. 5547, (November 2001), pp. 1713-1716, ISSN: 0193-4511 Courapied S., Cherier J., Vigneron A., Troadec M.B., Giraud S., Valo I., Prigent C., Gamelin

Dai Y. & Grant S. (2010). New insights into Checkpoint kinase 1 (Chk1) in the DNA damage

Danielsen J.M.R., Larsen D.H., Schou K.B., Freire R., Falck J., Bartek J. & Lukas J. (2009).

*Cancer*, Vol. 9, No. 1, (August 2010), pp. 205, ISSN: 1476-4598

Vol. 107, No. 31, (August 2010), pp. 13660-13665, ISSN: 0027-8424

ISSN: 0021-9258

Dordrecht, Holland

ISSN: 1474-175X

ISSN: 1046-2023

ISSN: 0036-8075

0027-5107

383, ISSN: 1078-0432

0021-9258

functional interaction between the human papillomavirus 16 transcription/replication factor E2 and the DNA damage response protein TopBP1. *The Journal of Biological Chemistry*, Vol. 277, No. 25, (June 2002), pp. 22297-22303,

*human diseases*, Nasheuer H.P., pp. 143-164, Springer, ISBN: 978-90-481-3470-0,

cycle control. *Nature Reviews Cancer*, Vol. 9, No. 11, (November 2009), pp. 785-797,

Reconsititution of RPA-covered single-stranded DNA activated ATR-Chk1 signaling. *Proceedings of the National Academy of Science of the United States of Americ*a,

checkpoint in a reconstituted system. *Methods*, Vol. 48, No. 1, (May 2009), pp. 3-7,

Mann M. (2009). Lysine acetylation targets protein complexes and co-regulates major cellular functions. *Science*, Vol. 325. No. 5942, (August 2009), pp. 834-840,

*Reviews Molecular Cell Biology*, Vol. 9, No. 8, (August 2008), pp. 616-627, ISSN: 1471-0072

radiation. *Mutation Research*, Vol. 704, No. 1-3, (April-June 2010), pp. 61-67, ISSN:

E., Coqueret O. & Barre B. (2010). Regulation of the Aurora-A gene following topoisomerase I inhibition: implication of the Myc transcription factor. *Molecular* 

response (DDR) signaling network: rationale for employing Chk1 inhibitors in cancer therapeutics. *Clinical Cancer Research*, Vol. 16, No. 2, (January 2010), pp. 376-

HCLK2 is required for activity of the DNA damage response kinase ATR. *The Journal of Biological Chemistry*, Vol. 284, No. 7, (February 2009), pp. 4140-4147, ISSN:

phosphorylation enhances the capacity for TopBP1 protein to activate ATR-ATRIP (Yoo et al., 2009). Yoo et al. (2009) showed that ATM can no longer interact with TopBP1 protein in NBS1-depleted egg extracts. Thus, the MRN complex helps to bridge ATM and TopBP1 together. ATM contributes to the activation of ATR through two collaborating mechanisms. First, ATM helps to create appropriate DNA structures that trigger activation of ATR. Second, ATM strongly stimulates the function of TopBP1 protein *via* its phosphorylation that directly carries out the ATR activation (Yoo et al., 2007).

### **8. Conclusion**

DNA is continuously exposed to a range of damaging agents, including reactive cellular metabolites, environmental chemicals, ionizing radiation and UV light. To prevent loss or incorrect transmission of genetic information and development of abnormalities and tumorigenesis all cells have evolved DNA damage response pathways to maintain their genome integrity. The DNA damage response involves the sensing of DNA damage signal to a network of cellular pathways, including cell cycle checkpoint, DNA repair and apoptosis. TopBP1 protein was first identified as an interacting partner for topoisomerase IIβ. This protein shares structural and functional similarities with BRCA1 and plays a critical role in the DNA damage response and checkpoint control. TopBP1 is essential for ATR activation in response to replication stress and UV-induced damage and also plays a direct role in the pathway that connects ATM to ATR in response to DSBs. The biological functions of TopBP1 protein, as well as its close relation with BRCA1 suggest a crucial role of TopBP1 in the maintenance of genome integrity and cell cycle regulation.

#### **9. References**


phosphorylation enhances the capacity for TopBP1 protein to activate ATR-ATRIP (Yoo et al., 2009). Yoo et al. (2009) showed that ATM can no longer interact with TopBP1 protein in NBS1-depleted egg extracts. Thus, the MRN complex helps to bridge ATM and TopBP1 together. ATM contributes to the activation of ATR through two collaborating mechanisms. First, ATM helps to create appropriate DNA structures that trigger activation of ATR. Second, ATM strongly stimulates the function of TopBP1 protein *via* its phosphorylation

DNA is continuously exposed to a range of damaging agents, including reactive cellular metabolites, environmental chemicals, ionizing radiation and UV light. To prevent loss or incorrect transmission of genetic information and development of abnormalities and tumorigenesis all cells have evolved DNA damage response pathways to maintain their genome integrity. The DNA damage response involves the sensing of DNA damage signal to a network of cellular pathways, including cell cycle checkpoint, DNA repair and apoptosis. TopBP1 protein was first identified as an interacting partner for topoisomerase IIβ. This protein shares structural and functional similarities with BRCA1 and plays a critical role in the DNA damage response and checkpoint control. TopBP1 is essential for ATR activation in response to replication stress and UV-induced damage and also plays a direct role in the pathway that connects ATM to ATR in response to DSBs. The biological functions of TopBP1 protein, as well as its close relation with BRCA1 suggest a crucial role of TopBP1

Araki H., Leem S.H., Phongdara A. & Sugino A. (1995). Dpb11, which interacts with DNA

*Cellular Biology*, Vol. 27, No. 9, (May 2007), pp. 3367-3377, ISSN: 0270-7306 Bang S.W., Ko M.J., Kang S., Kim G.S., Kang D., Lee J.H. & Hwang D.S. (2011). Human TopBP1

Beausoleil S.A., Villen J., Gerber S.A., Rush J. & Gygi S.P. (2006). A probability-based

Binz S.K., Sheehan A.M. & Wold M.S. (2004). Replication protein A phosphorylation and the

Bolderson E., Tomimatsu N., Richard D.J., Boucher D., Kumar R., Pandita T.K., Burma S. &

*Research*, Vol. 317, No. 7, (April 2011), pp. 994-1004, ISSN: 0014-4827

polymerase II (ε) in *Sacharomyces cerevisiae*, has a dual role in S-phase progression and at a cell cycle checkpoint. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 92, No. 25, (December 1995), pp. 11791-11795, ISSN: 0027-8424 Ball H.L., Ehrhardt M.R., Mordes D.A., Glick G.G., Chazin W.J. & Cortez D. (2007). Function

of a conserved checkpoint recruitment domain in ATRIP proteins. *Molecular and* 

localization to the mitotic centrosome mediates mitotic progression. *Experimental Cell* 

approach for high-throughput protein phosphorylation analysis and site localization. *Nature Biotechnology*, Vol. 24, No. 10, (October 2006), pp. 1285-1292,

cellular response to DNA damage. *DNA Repair*, Vol. 3, No. 8-9, (August-September

Khanna K.K. (2009). Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks. *Nucleic Acids Research*, Vol. 38,

that directly carries out the ATR activation (Yoo et al., 2007).

in the maintenance of genome integrity and cell cycle regulation.

**8. Conclusion** 

**9. References** 

ISSN: 1087-0156

2004), pp. 1015-1024, ISSN: 1568-7856

No. 6, (December 2009), pp. 1821-1831, ISSN: 0305-1048


TopBP1 in DNA Damage Response 299

Herold S., Wenzel M., Beuger V., Frohme C., Beul D., Hillukkala T., Syvaoja J., Saluz H.P.,

Hiom K. (2010). Coping with DNA double strand breaks. *DNA Repair*, Vol. 9, No. 12,

Honda Y., Tojo M., Matsuzaki K., Anan T., Matsumoto M., Ando M., Saya H. & Nakao M.

Horton J.K., Stefanick D.F., Zeng J.Y., Carrozza M.J. & Wilson S.H. (2011). Requirement for

Houtgraaf J.H., Versmissen J. & van der Giessen W.J. (2006). A concise review of DNA

*Medicine*, Vol. 7, No. 3, (July-September 2006), pp. 165-172, ISSN: 1553-8389 Huo Y., Bai L., Xu M. & Jiang T. (2010). Crystal structure of the N-terminal region of human

Jazayeri A., Balestrini A., Garner E., Haber J.E. & Costanzo V. (2008). Mre11-Rad50-Nbs1-

Jeon Y., Ko E., Lee K.Y., Ko M.J, Park S.Y., Kang J., Jeon C.H., Lee H. & Hwang D.S. (2011).

Jungmichel S. & Stucki M. (2010). MDC1: The art of keeping things in focus. *Chromosoma*,

Kanaar R. & Wyman C. (2008). DNA repair by the MRN complex: break it to make it. *Cell*,

Karppinen S.M., Erkko H., Reini K., Pospiech H., Heikkinen K., Rapakko K., Syvaoja J.E. &

*Repair*, Vol. 8, No. 9, (September 2009), pp. 1139-1152, ISSN: 1568-7856 Kim J.E., McAvoy S.A., Smith D.I. & Chen J. (2005). Human TopBP1 ensures genome

Press, ISBN: 978-1-60761-177-6, Totowa, New Jersey, USA

Vol. 119, No. 4, (August 2010), pp. 337-349, ISSN: 0009-5915

Vol. 135, No. 1, (October 2008), pp. 14-16, ISSN: 0092-8674

(December 2005), pp. 10907-10915, ISSN: 0270-7306

(February 2011), pp. 5414-5422, ISSN: 0021-9258

2002), pp. 509-521, ISSN: 1097-2765

1568-7856

4189

(December 2010), pp. 1256-1263, ISSN: 1568-7856

Haenel F. & Eilers M. (2002). Negative regulation of the mammalian UV response by Myc through association with Miz-1. *Molecular Cell*, Vol. 10, No. 3, (September

(2002). Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response. *The Journal of Biological Chemistry*, Vol. 277, No. 5, (February 2002), pp. 3599-3605, ISSN: 0021-9258

NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition. *DNA Repair*, Vol. 10, No. 2, (February 2011), pp. 225-234, ISSN:

damage checkpoints and repair in mammalian cells. *Cardiovascular Revascularization* 

Topoisomerase IIβ binding protein 1. *Biochemical and Biophysical Research Communications*, Vol. 401, No. 3, (October 2010), pp. 401-405, ISSN: 0006-291X Hurley P.J. & Bunz F. (2009). Distinct pathways involved in S-phase checkpoint control, In:

*Checkpoint Controls and Targets in Cancer Therapy*, Siddik Z.H., pp. 27-36, Humana

dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity. *EMBO Journal*, Vol. 27, No. 14, (July 2008), pp. 1953-1962, ISSN: 0261-

TopBP1 deficiency causes an early embryonic lethality and induces cellular senescence in primary cells. *The Journal of Biological Chemistry*, Vol. 286, No. 7,

Winqvist R. (2006). Identification of a common polymorphism in the TopBP1 gene associated with hereditary susceptibility to breast and ovarian cancer. *European Journal of Cancer*, Vol. 42, No. 15, (October 2006), pp. 2647-2652, ISSN: 0959-8049 Kerzendorfer C. & O'Driscoll M. (2009). Human DNA damage response and repair deficiency

syndromes: Linking genomic instability and cell cycle checkpoint proficiency. *DNA* 

integrity during normal S phase. *Molecular and Cellular Biology*, Vol. 25, No. 24,


Delacroix S., Wagner J.M., Kobayashi M., Yamamoto K. & Karnitz L.M. (2007). The Rad9-

Dijkstra K.K., Blanchetot C. & Boonstra J. (2009). Evasion of G1 checkpoint in cancer, In:

Essers J., Vermeulen W. & Houtsmeller A.B. (2006). DNA damage repair: anytime,

Fannig E., Klimovich V. & Nager A.R. (2006). A dynamic model for replication protein A

Garcia V., Furuya K. & Carr A.M. (2005). Identification and functional analysis of TopBP1

Gburcik V., Bot N., Maggiolini M. & Picard D. (2005). SPBP is a phosphoserine-specific

Germann S.M., Oestergaard V.H., Haas C., Salis P., Motegi A. & Lisby M. (2011).

Glover J.N.M. (2006). Insights into the molecular basis of human hereditary breast cancer

Glover J.N.M., Williams R.S. & Lee M.S. (2004). Interactions between BRCT repeats and

Going J.J., Nixon C., Dornan E.S., Boner W., Donaldson M.M. & Morgan I.M. (2007).

Greer D.A., Besley B.D.A., Kennedy K.B. & Davey S. (2003). hRad9 rapidly binds DNA

Hashimoto Y., Tsujimura T., Sugino A. & Takisawa H. (2006). The phosphorylated C-

Herold S., Hock A., Herkert B., Berns K., Mullenders J., Beijersbergen R., Bernards R. &

Press, ISBN: 978-1-60761-177-6, Totowa, New Jersey, USA

15, (August 2006), pp. 4126-4137, ISSN: 0305-1048

2005), pp. 3421-3430, ISSN: 0270-7306

2006), pp. 89-93, ISSN: 1389-9600

(November 2004), pp. 579-585, ISSN: 0968-0004

16, (August 2003), pp. 4829-4835, ISSN: 0008-5472

(March 2007), pp. 418-424, ISSN: 0309-0167

*Development*, Vol. 21, No. 12, (June 2007), pp. 1472-1477, ISSN: 0890-9369 Dephoure N., Zhou C., Villen J., Beausoleil S.A., Bakalarski C.E., Elledge S.J. & Gygi

2008), pp. 10762-10767, ISSN: 0027-8424

ISSN: 0955-0674

ISSN: 1568-7856

224, ISSN: 1568-7856

0261-4189

Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling *via* TopBP1. *Genes and* 

S.P.(2008). A quantitative atlas of mitotic phosphorylation. *Proceedings of the National Academy of Science of the United States of America*, Vol. 105, No. 31, (August

*Checkpoint Controls and Targets in Cancer Therapy*, Siddik Z.H., pp. 3-26, Humana

anywhere? *Current Opinion in Cell Biology*, Vol. 18, No. 3, (June 2006), pp. 240-246,

(RPA) function in DNA processing pathways. *Nucleic Acids Research*, Vol. 34, No.

and its homologs. *DNA Repair*, Vol. 4, No. 11, (November 2005), pp. 1227-1239,

repressor of estrogen receptor α. *Molecular and Cellular Biology*, Vol. 25, No. 9, (May

Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination. *DNA Repair*, Vol. 10, No. 2, (February 2011), pp. 210-

from studies of the BRCA1 BRCT domain. *Familial Cancer*, Vol. 5, No. 1, (March

phosphoproteins: tangled up in two. *Trends in Biochemical Sciences*, Vol. 29, No. 11,

Aberrant expression of TopBP1 in breast cancer. *Histopathology*, Vol. 50, No. 4,

containing double-strand breaks and is required for damage-dependent topoisomerase IIβ binding protein 1 focus formation. *Cancer Research*, Vol. 63, No.

terminal domain of *Xenopus* Cut5 directly mediates ATR-dependent activation of Chk1. *Genes to Cells*, Vol. 11, No. 9, (September 2006), pp. 993-1007, ISSN: 1356-9597

Eilers M. (2008). Miz1 and HectH9 regulate the stability of the checkpoint protein, TopBP1. *EMBO Journal*, Vol. 27, No. 21, (November 2008), pp. 2851-2861, ISSN:


TopBP1 in DNA Damage Response 301

Medhurst A.L., Warmerdam D.O., Akerman I., Verwayen E.H., Kanaar R., Smits V.A.J. &

Mordes D.A. & Cortez D. (2008). Activation of ATR and related PIKKs. *Cell Cycle*, Vol. 7, No.

Mordes D.A., Glick G.G., Zhao R. & Cortez D. (2008). TopBP1 activates ATR through ATRIP

Morishima K., Sakamoto S., Kobayashi J., Izumi H., Suda T., Matsumoto Y., Tauchi H., Ide

*Veterinary Journal*, Vol. 179, No. 3, (March 2009), pp. 422-429, ISSN: 1090-0233 Muniandy P., Liu J., Majumdar A., Liu S. & Seidman M.M. (2010). DNA interstrand cross

*Molecular Biology*, Vol. 45, No. 1, (February 2010), pp. 23-49, ISSN: 1040-9238 Nakanishi M. (2009). Chromatin modifications and orchestration of checkpoint response in

Nakanishi M., Niida H., Murakami H. & Shimada M. (2009). DNA damage responses in skin

O'Driscoll M. & Jeggo P.A. (2006). The role of double-strand break repair – insights from

Ogiwara H., Ui A., Onoda F., Tada S., Enomoto T. & Seki M. (2006). Dpb11, the pudding yeast

Pallis A.G. & Karamouzis M.V. (2010). DNA repair pathways and their implication in cancer

Parrilla-Castellar E.R. & Karnitz L.M. (2003). Cut5 is required for the binding of Atr and

Vol. 281, No. 51, (December 2006), pp. 39517-39533, ISSN: 0021-9258

Humana Press, ISBN: 978-1-60761-177-6, Totowa, New Jersey, USA

21, No. 1, (January 2006), pp. 3-9, ISSN: 0267-8357

(September 2010), pp. 591-601, ISSN: 0344-0338

18, (September 2008), pp. 2809-2812, ISSN: 1551-4005

ISSN: 0193-4511

ISSN: 1471-0056

685, ISSN: 0167-7659

3933-3940, ISSN: 0021-9533

pp. 1478-1489, ISSN: 0890-9369

S.J. (2007). ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. *Science*, Vo. 316, No. 5828, (May 2007), pp. 1160-1166,

Lakin N.D. (2008). ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage*. Journal of Cell Science*, Vol. 121, No. 23, (December 2008), pp.

and a PIKK regulatory domain. *Genes and Development*, Vol. 22, No. 11, (June 2008),

H., Komatsu K. & Matsuura S. (2007). TopBP1 associates with NBS1 and is involved in homologous recombination repair. *Biochemical and Biophysical Research Communications*, Vol. 362, No. 4, (November 2007), pp. 872-879, ISSN: 0006-291X Morris J.S., Nixon C., King O.J.A., Morgan I.M. & Philbey A.W. (2009). Expression of TopBP1

in canine mammary neoplasia in relation to histological type, Ki67, ERα and p53. *The* 

link repair in mammalian cells: step by step. *Critical Reviews in Biochemistry and* 

cancer, In: *Checkpoint Controls and Targets in Cancer Therapy*, Siddik Z.H., pp. 83-94,

biology – Implication in tumor prevention and aging acceleration. *Journal of Dermatological Science*, Vol. 56, No. 2, (November 2009), pp. 76-81, ISSN: 0923-1811 Niida H. & Nakanishi M. (2006). DNA damage checkpoints in mammals. *Mutagenesis*, Vol.

human genetics. *Nature Reviews Genetics*, Vol. 7, No. 1, (January 2006), pp. 45-54,

homolog of TopBP1, functions with the checkpoint clamp in recombination repair. *Nucleic Acids Research*, Vol. 34, No. 11, (July 2006), pp. 3389-3398, ISSN: 0305-1048 Olson E., Nievera C.J., Klimovivh V., Fanning E. & Wu X. (2006). RPA2 is a direct downstream

target for ATR to regulate the S-phase checkpoint. *The Journal of Biological Chemistry*,

treatment. *Cancer and Metastasis Reviews*, Vol. 29, No. 4, (September 2010), pp. 677-

DNA polymerase α to genotoxin-damaged chromatin. *The Journal of Biological Chemistry*, Vol. 278, No. 46, (November 2003), pp. 45507-45511, ISSN: 0021-9258 Poehlmann A. & Roessner A. (2010). Importance of DNA damage checkpoints in the

pathogenesis of human cancers. *Pathology Research and Practice*, Vol. 206, No. 9,


Kumagai A., Kim S.M. & Dunphy W.G. (2004). Claspin and the activated from of ATR-

Kumagai A., Lee J., Yoo H.Y. & Dunphy W.G. (2006). TopBP1 activates the ATR-ATRIP complex. *Cell*, Vol. 124, No. 5, (March 2006), pp. 943-955, ISSN: 0092-8674 Lee J. & Dunphy W.G. (2010). Rad17 plays a central role in establishment of the interaction

Lee J., Kumagai A. & Dunphy W.G. (2007). The Rad9-Hus1-Rad1 checkpoint clamp

Lelung C.C.Y., Kellogg E., Kuhnert A., Hanel F., Baker D. & Glover J.N.M. (2010). Insights

Liu K., Bellam N., Lin H.Y., Wang B., Stockard C.R., Grizzle W.E. & Lin W.C. (2009).

Liu K., Paik J.C., Wang B., Lin F.T. & Lin W.C. (2006a). Regulation of TopBP1

Liu S., Bekker-Jensen S., Mailand N., Lukas C., Bartek J. & Lukas J. (2006b). Claspin operates

Liu K., Lin F.T., Ruppert J.M. & Lin W.C. (2003). Regulation of E2F1 by BRCT domain-

Lopez-Contreras A.J. & Fernandez-Capetillo O. (2010). The ATR barrier to replication-born

Lyngso C., Bouteiller G., Damgaard C.K., Ryom D., Sanchez-Munoz S., Norby P.L., Bonven

Majka J., Binz S.K., Wold M.S. & Burgers P.M. (2006). Replication protein A directs loading

*Chemistry*, Vol. 276, No. 32, (August 2001), pp. 30399-30406, ISSN: 0021-9258 Matsuoka S., Ballif B.A., Smogorzewska A., McDonald III E.R., Hurov K.E., Luo J.,

279, No. 48, (November 2004), pp. 49599-49608, ISSN: 0021-9258

282, No. 38, (September 2007), pp. 28036-28044, ISSN: 0021-9258

(October 2006), pp. 4795-4807, ISSN: 0261-4189

2003), pp. 3287-3304, ISSN: 0270-7306

2000), pp. 26144-26149, ISSN: 0021-9258

0270-7306

0890-9369

1568-7856

ATRIP collaborate in the activation of Chk1. *The Journal of Biological Chemistry*, Vol.

between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks. *Molecular Biology of the Cell*, Vol. 21, No. 6, (March 2010), pp. 926-935, ISSN: 1059-1524

regulates interaction of TopBP1 with ATR. *The Journal of Biological Chemistry*, Vol.

from the crystal structure of the sixth BRCT domain of topoisomerase IIβ binding protein 1. *Protein Science*, Vo. 19, No. 1, (January 2010), pp. 162-167, ISSN: 0961-8368

Regulation of p53 by TopBP1: a potential mechanism for p53 inactivation in cancer, *Molecular and Cellular Biology*, Vol. 29, No. 10, (May 2009), pp. 2673-2693, ISSN:

oligomerization by Akt/PKB for cell survival. *EMBO Journal*, Vol. 25, No. 20,

downstream of TopBP1 to direct ATR signaling towards Chk1 activation. *Molecular and Cellular Biology*, Vol. 26, No. 16, (August 2006), pp. 6056-6064, ISSN: 0270-7306 Liu K., Luo Y., Lin F.T. & Lin W.C. (2004). TopBP1 recruits Brg1/Brm to repress E2F1-

induced apoptosis, a novel pRb-independent and E2F1-specific control for cell survival. *Genes and Development*, Vol. 18, No. 6, (March 2004), pp. 673-686, ISSN:

containing protein TopBP1. *Molecular and Cellular Biology*, Vol. 23, No. 9, (May

DNA damage. *DNA Repair*, Vol. 9, No. 12, (December 2010), pp. 1249-1255, ISSN:

B.J. & Jorgensen P. (2000). Interaction between the transcription factor SPBP and the positive cofactor RNF4. *The Journal of Biological Chemistry*, Vol. 275, No. 34, (August

of the DNA damage checkpoint clamp to 5'-DNA junctions. *The Journal of Biological Chemistry*, Vol. 281, No. 38, (September 2006), pp. 27855-27861, ISSN: 0021-9258 Makiniemi M., Hillukkala T., Tuusa J., Reini K., Vaara M., Huang D., Pospiech H., Majuri I.,

Westerling T., Makela T.P. & Syvaoja J.E. (2001). BRCT domain-containing protein TopBP1 functions in DNA replication and damage response. *The Journal of Biological* 

Bakalarski C.E., Zhao Z., Solimini N., Lerenthal Y., Shiloh Y., Gygi S.P. & Elledge

S.J. (2007). ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. *Science*, Vo. 316, No. 5828, (May 2007), pp. 1160-1166, ISSN: 0193-4511


TopBP1 in DNA Damage Response 303

Shiotani B. & Zou L. (2009). Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks. *Molecular Cell*, Vol. 33, No. 5, (March 2009), pp. 547-558, ISSN: 1097-2765 Sjottem E., Rekdal C., Svineng G., Johansen S.S., Klenow H., Uglehus R.D. & Johansen T.

Smits V.A.J., Warmerdam D.O., Martin Y. & Freire R. (2010). Mechanisms of ATR-mediated

Sokka M., Parkkinen S., Pospiech H. & Syvaoja J.E. (2010), Function of TopBP1 in genome

Su Y., Meador J.A., Geard C.R. & Balajee A.S. (2010). Analysis of ionizing radiation-induced

Takeishi Y., Ohashi E., Ogawa K., Masai H., Obuse C. & Tsurimoto T. (2010). Casein kinase

Taricani L. & Wang T.S.F. (2006). Rad4TopBP1, a scaffold protein, plays separate roles in DNA

Van C., Yan S., Michael W.M., Waga S. & Cimprich K.A. (2010). Continued primer synthesis

Van Hatten R.A., Tutter A.V., Holway A.H., Khederian A.M., Walter J.C. & Michael W.M.

Wang B., Malik R., Nigg E.A. & Korner R. (2008). Evaluation of the low-specificity protease

Wanzel M., Herold S. & Eilers M. (2003). Transcriptional repression by Myc. *Trends in Cell* 

Warmerdam D.O. & Kanaar R. (2010). Dealing with DNA damage: Relationships between

Williams R.S., Williams J.S. & Tainer J.A. (2007). Mre11-Rad50-Nbs1 is a keystone complex

Wollmann Y., Schmidt U., Wieland G.D., Zipfel P.F., Saluz H.P. & Hanel F. (2007). The DNA

Woodhouse B.C. & Dianov G.L. (2008). Poly ADP-ribose polymerase-1: An international

*Cell*, Vol. 17, No. 8, (August 2006), pp. 3456-3468, ISSN: 1059-1524

*Biology*, Vol. 189, No. 2, (April 2010), pp. 233-246, ISSN: 0021-9525

*Biology*, Vol. 13, No. 3, (March 2003), pp. 146-150, ISSN: 0962-8924

(October 2007), pp. 6648-6662, ISSN: 0305-1048

2010), pp. 761-771, ISSN: 1356-9597

2002), pp. 541-547, IISN: 0021-9525

2010), pp. 2-11, ISSN: 0027-5107

2007), pp. 171-182, ISSN: 0730-2312

509-520, ISSN: 0829-8211

1568-7856

24, (December 2008), pp. 9536-9533, ISSN: 0003-2700

Springer, ISBN: 978-90-481-3470-0, Dordrecht, Holland

ISSN: 1093-4715

(2007). The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription. *Nucleic Acids Research*, Vol. 35, No. 19,

checkpoint signaling. *Frontiers in Bioscience*, Vol. 15, No. 3, (June 2010), pp. 840-853,

stability, In: *Genome stability and human diseases*, Nasheuer H.P., pp. 119-141,

DNA damage and repair in three-dimensional human skin model system. *Experimental Dermatology*, Vol. 19, No. 8, (August 2010), pp. e16-22, ISSN: 0906-6705

2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1. *Genes to Cells*, Vol. 15, No. 7, (June

damage and replication checkpoints and DNA replication. *Molecular Biology of the* 

at stalled replication forks contributes to checkpoint activation. *The Journal of Cell* 

(2002). The *Xenopus* Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. *The Journal of Cell Biology*, Vol. 159, No. 4, (November

elastase for large-scale phosphoproteome analysis. *Analytical Chemistry*, Vol. 80, No.

checkpoint and repair pathways. *Mutation Research*, Vol. 704, No. 1-3, (April-June

connecting DNA repair machinery, double-strand break signaling, and the chromatin template. *Biochemistry and Cell Biology*, Vol. 85, No. 4, (August 2007), pp.

topoisomerase IIβ binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1). *Journal of Cellular Biochemistry*, Vol. 102, No. 1, (September

molecule of mystery. *DNA Repair*, Vol. 7, No. 7, (July 2008), pp. 1077-1086, ISSN:


Pommier Y., Leo E., Zhang H.L. & Marchand C. (2010). DNA topoisomerases and their

Poznic M. (2009). Retinoblastoma protein: a central processing unit. *Journal of Biosciences*,

Rappas M., Oliver A.W. & Pearl L.H. (2011). Structure and function of the Rad9-binding

Rastogi R.P., Richa, Kumar A., Tyagi M.B. & Sinha R.P. (2010). Molecular mechanisms of

Reinhardt H.C. & Yaffe M.B. (2009). Kinases that control the cell cycle in response to DNA

Reinhardt H.C., Hasskamp P., Schmedding I., Morandell S., van Vugt M.A.T.M., Wang X.Z.,

Reini K., Uitto L., Perera D., Moens P.B., Freire R. & Syvaoja J.E. (2004). TopBP1 localises to

Rekdal C., Sjottem E. & Johansen T. (2000). The nuclear factor SPBP contains different

Rodriguez-Bravo V., Guaita-Esteruelas S., Salvador N., Bachs O. & Agell N. (2007). Different S/M

Rupnik A., Lownde N. & Grenon M. (2010). MRN and the race to the break. *Chromosoma*,

Sansam C.L., Cruz N.M., Danielian P.S., Amsterdam A., Lau M.L., Hopkins N. & Lees J.A.

Scheffner M. & Staub O. (2007). HECT E3s and human disease. *BioMed Central Biochemistry*,

Schmidt U., Wollmann Y., Franke C., Grosse F., Saluz H.P. & Hanel F. (2008).

278, No. 52, (December 2003), pp. 52914-52918, ISSN: 0021-9258

*Biology*, Vol. 15, No. 6, (June 1995), pp. 3164-3170, ISSN: 0270-7306

Vol. 119, No. 2, (April 2010), pp. 115-135, ISSN: 0009-5915

Vol. 8, Suppl. 1, (November 2007), pp. S6, ISSN: 1471-2091

5, (May 2010), pp. 421-433, ISSN: 1074-5521

Vol. 34, No. 2, (June 2009), pp. 305-312, ISSN: 0250-5991

39, No. 1, (January 2011), pp. 313-324, ISSN: 0305-1048

2010:592980, (December 2010), ISSN: 2090-0201

(April 2009), pp. 245-255, ISSN: 0955-0674

(October 2010), pp. 34-49, ISSN: 1097-2765

ISSN: 0021-9258

1551-4005

No. 7, (May 2004), pp. 323-330, ISSN: 0009-5915

poisoning by anticancer and antibacterial drugs. *Chemistry and Biology*, Vol. 17, No.

region of the DNA-damage checkpoint adaptor TopBP1. *Nucleic Acids Research*, Vol.

ultraviolet radiation-induced DNA damage and repair. *Journal of Nucleic Acids*, Vol.

damage: Chk1, Chk2, and MK2. *Current Opinion in Cell Biology*, Vol. 21, No. 2,

Linding R., Ong S.E., Weaver D., Carr S.A. & Yaffe M.B. (2010). DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization. *Molecular Cell*, Vol. 40, No. 1,

centrosomes in mitosis and to chromosome cores in meiosis. *Chromosoma*, Vol. 112,

functional domains and stimulates the activity of various transcriptional activators. *The Journal of Biological Chemistry*, Vol. 275, No. 51, (December 200), pp. 40288-40300,

checkpoint responses of tumor and non-tumor cell lines to DNA replication inhibition. *Cancer Research*, Vol. 67, No. 24, (December 2007), pp. 11648-11656, ISSN: 0008-5472 Rogriguez M., Yu X., Chen J. & Songyang Z. (2003). Phosphopeptide binding specificities of

BRCA1 COOH-terminal (BRCT) domains. *The Journal of Biological Chemistry*, Vol.

(2010). A vertebrate gene, *ticrr*, is an essential checkpoint and replication regulator. *Genes and Development*, Vol. 24, No. 2, (January 2010), pp. 183-194, ISSN: 0890-9369 Sanz L., Moscat J. & Diaz-Meco M. (1995). Molecular characterization of a novel

transcription factor that controls stromelysin expression. *Molecular and Cellular* 

Characterization of the interaction between the human DNA topoisomerase IIβbinding protein (TopBP1) and the cell division cycle 45 (Cdc45) protein. *The Biochemical Journal*, Vol. 409, No. 1, (January 2008), pp. 169-177, ISSN: 0264-6021 Scorah J. & McGowan C.H. (2009). Claspin and Chk1 regulate replication fork stability by

different mechanisms. *Cell Cycle*, Vol. 8, No. 7, (April 2009), pp. 1036-1043, ISSN:


**16** 

*Canada* 

**Post-Meiotic DNA Damage** 

Guylain Boissonneault et al.\*

 *Université de Sherbrooke* 

**and Response in Male Germ Cells** 

*Department of Biochemistry, Faculty of Medicine and Health Sciences* 

Spermatids are haploid cells that differentiate into spermatozoa and may be considered as an interesting model of DNA damage response and repair. Key features, such a unique set of chromosomes, radioresistance to apoptosis, the presence of known end-joining DNA repair pathways and an underlying prerogative to limit the transmission of any mutation to the next generation, make them a unique cell type to provide new insights on similar pathways in somatic cells. Although DNA damage signaling and repair mechanisms have been extensively studied during meiosis, the contribution of post-meiotic germ cells to the genetic integrity of the male gamete have been overlooked. In this chapter we present clear evidences that the haploid phase of spermatogenesis, termed spermiogenesis, may represent an even greater challenge for the maintenance of the genetic integrity of the male gamete. Since transient DNA strand breaks are intrinsic to the differentiation program of spermatids (Leduc et al., 2008a; Marcon and Boissonneault, 2004), a better understanding of DNA repair pathways involved may shed some light on their potential contribution to male-driven *de novo* mutations and eventually to some unresolved cases of male infertility. This chapter will mainly focus on DNA breaks occurring in the post-meiotic phase of the spermatogenesis

In most mammals, testes are found in the scrotum and are maintained at lower temperature (2-8°C) than the core body (Harrison and Weiner, 1949; Setchell, 1998). In fact, spermatogenesis is known to work better at lower temperature and it was shown that fertility declines with scrotal hyperthermia. For example, higher scrotal temperature due to fever or lifestyle correlates with decreased semen quality in humans (reviewed in Jung and

To support germ cells in their development, Sertoli cells are located at the basal lamina, throughout the seminiferous tubules (Russell, 1990). They provide nutrients and essential

Frédéric Leduc, Geneviève Acteau, Marie-Chantal Grégoire, Olivier Simard, Jessica Leroux, Audrey

*Department of biochemistry, Faculty of medicine and health sciences, Université de Sherbrooke, Canada* 

**1. Introduction** 

and how germ cells deal with it.

Carrier-Leclerc and Mélina Arguin

**2. Spermatogenesis** 

Schuppe, 2007).

 \*

