**5. TopBP1 as multifunctional protein**

TopBP1 protein has been proposed as a transcriptional repressor of E2F1 and transcriptional co-activator with HPV16 E2 (Liu et al., 2004; Wright et al., 2006; Yoshida & Inoue, 2004). The E2F transcription factors E2F1 to E2F6 bind to E2F sites in promoters and regulate the expression of a large array of genes that encode proteins important for DNA replication and cell cycle progression. In response to growth signals, activated G1 cycline-dependent kinase phosphorylate retinoblastoma protein (Rb) and release E2F from Rb binding. This event is critical in controlling G1/S transition. Among the E2F family members, E2F1, E2F2 and E2F3 are transcriptional activators and are induced in response to growth stimulation, with peak accumulation at G1/S. Together, they are essential for cellular proliferation since a combined mutation of E2F1, E2F2 and E2F3 completely blocks cellular proliferation. In contrary, E2F4 and E2F5 act mainly as transcriptional repressors (Chen et al., 2009; Liu et al., 2003; Poznic, 2009). TopBP1 protein interacts with E2F1 through the sixth BRCT motif of TopBP1 and N terminus of E2F1 (Fig. 2) (Lelung et al., 2010; Liu et al., 2003). This interaction is induced by ATM-mediated phosphorylation of E2F1 at Ser31 during DNA damage. By this interaction, the transcriptional activity of E2F1 is repressed and E2F1 is recruited to DNA damage induced nuclear foci (Liu et al., 2003). Moreover, the interaction between TopBP1 protein and E2F1, as well as the repression of E2F1 activity, are specific for E2F1 but are not seen in E2F2, E2F3 and E2F4, suggesting that TopBP1 is E2F1 exclusive regulator (Liu et al., 2004). Liu et al. (2004) showed that E2F1 is also regulated by a novel Rb-independent mechanism, in which TopBP1 protein recruits Brg1/BRM (Brahma-related gene 1/Brahma protein), a central subunit of the SWI/SNF (SWItch/sucrose nonfermentable) chromatin modeling complex, to specifically inhibit E2F1 transcriptional activity. This regulation appeared to be critical for E2F1-dependent apoptosis control during S phase and DNA damage. On the other hand, TopBP1 is induced by E2F1 and interacts with E2F1 during G1/S transition. Thus, E2F1 and TopBP1 form a feedback regulation to prevent apoptosis during DNA replication (Liu et al., 2003).

Human papillomaviruses (HPVs) are causative agents in a number of human diseases the most common of which is cervical cancer. More than 95% of cervical carcinomas harbor HPV sequences and HPV16 is most frequently detected. The HPV16 E2 protein is a 43 kDa phosphoprotein that binds as a homodimer to 12 bp palindromic DNA sequences in the transcriptional control region of the viral genome. After binding, E2 can either upregulate or repress transcription from the adjacent promoter depending on cell type and protein levels and this regulation controls the expression of viral oncoproteins E6 and E7. The carboxylterminal portion of TopBP1 interacts with E2 and TopBP1 protein can enhance the ability of E2 to activate transcription and replication (Fig. 2) (Boner et al., 2002).

TopBP1 protein also interacts with SPBP (stromelysin-1 platelet-derived growth factor (PDGF) responsive element binding protein) and enhances the transcriptional activity of Ets1 on the *Myc* and *MMP-3* promoters *in vitro* and *in vivo* (Sjottem et al., 2007). This

TopBP1 in DNA Damage Response 289

Miz-1 from the complex with TopBP1 (Wollmann et al., 2007; Yamane et al., 1997; Yamane &

Site(s) Modification Enzyme Reference

region phosphorylation c-Abl Zeng et al., 2005 S214 phosphorylation ATM/ATR Matsuoka et al., 2007 S492 phosphorylation ATM Sokka et al., 2010 S405 phosphorylation ATM/ATR Matsuoka et al., 2007 S409 phosphorylation ATM/ATR Matsuoka et al., 2007 S554 phosphorylation ATM Sokka et al., 2010 K581 acetylation N/D Choudhary et al., 2010 S766 phosphorylation ATM Sokka et al., 2010 S805 phosphorylation N/D Beausoleil et al., 2006;

T848 phosphorylation N/D Dephoure et al., 2008 S860 phosphorylation N/D Dephoure et al., 2008 S861 phosphorylation N/D Dephoure et al., 2008 S864 phosphorylation N/D Dephoure et al., 2008

T975 phosphorylation ATM/ATR Matsuoka et al., 2007 S1002 phosphorylation N/D Dephoure et al., 2008;

S1051 phosphorylation ATM/ATR Matsuoka et al., 2007 T1062 phosphorylation ATM Sokka et al., 2010 T1086 phosphorylation ATM/ATR Matsuoka et al., 2007 S1138 phosphorylation ATM Yoo et al., 2007 S1159 phosphorylation Akt Liu et al., 2006 Table 1. Post-translation modifications of the human TopBP1 protein (N/D – not determined) Apart from the mentioned above ADP-ribosylation, TopBP1 undergoes other posttranslational modifications, such as acetylation and phosphorylation (Table 1). Lysine acetylation is a reversible post-translational modification, which neutralizes the positive charge of this amino acid changing protein function. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport and actin nucleation. Acetylation of TopBP1 protein occurs at position 581 but the exact role of this modification remains to be

TopBP1 is a phosphoprotein and is phosphorylated in response to DNA damage (Makiniemi et al., 2001; Yamane et al., 2003). After DNA damage, TopBP1 protein localizes at IR-induced nuclear foci and is phosphorylated by ATM kinase (Yamane et al.,

S888 phosphorylation N/D

(BRCT6) ADP-ribosylation PARP-1

Wang et al., 2008

Beausoleil et al., 2006; Dephoure et al., 2008; Wang et al., 2008

Wollmann et al., 2007; Yamane et al., 1997; Yamane & Tsuruo, 1999

Wang et al., 2008

Tsuruo, 1999).

Y in BRCT1-4

900-991

resolved (Choudhary et al., 2010).

interaction is mediated by ePHD (extended plant homeodomain) domain of SPBP and the BRCT6 domain of TopBP1 (Sjottem et al., 2007). SPBP a 220 kDa ubiquitously expressed nuclear protein is shown to intensify or repress the transcriptional activity. Originally SPBP was identified as a protein involved in transcriptional activation of matrix metalloproteinase-3 (MMP3), stromelysin-1 promoter *via* the specific sequence element SPRE (stromelysin-1 PDGF responsive element) (Rekdal et al., 2000; Sonz et al., 1995). Later SPBP was found to act as a transcriptional coactivator since it enhanced the transcriptional activity of the positive cofactor and RING finger protein SNURF/RNF4 (small nuclear RING finger protein/RING finger protein 4) and of certain transcription factors, such as Sp1 (specificity protein 1), Ets (E-twenty-six specific), Pax6 (paired box gene 6) and Jun (Lyngso et al., 2000; Rekdal et al., 2000; Sjottem et al., 2007). On the other hand, SPBP appears to act as phosphoserine-specific repressor of estrogen receptor α (ERα) (Gburick et al., 2005; Sjottem et al., 2007).

In unstressed cells TopBP1 protein associates with Miz-1 (Myc interacting zinc finger protein 1). BRCT6 and BRCT7 of TopBP1 are required and largely sufficient to mediate the interaction with Miz-1 (Fig. 2) (Herold et al., 2002, 2008; Wenzel et al., 2003). This zinc finger protein that contains an amino-terminal POZ (poxvirus and zinc finger) was initially described as a protein that interacts with C terminus of Myc oncoprotein (Courapied et al., 2010; Herold et al., 2008). Miz-1 protein activates transcription of genes encoding the cell cycle inhibitors p15INK46 and p21Cip1, leading to cell cycle arrest. Miz-1 can also repress transcription when it forms complexes with Myc and other transcription factors (Herold et al., 2002, 2008; Wenzel et al., 2003). In response to UV irradiation Miz-1 is released from an inhibitory complex formed with TopBP1 and binds to the start site of *p21Cip1* promoter. Thus the dissociation of TopBP1 from Miz-1 may facilitate the induction of *p21Cip1* (Herold et al., 2002, 2008; Wenzel et al., 2003). On the other hand, Miz-1 is required for the binding of TopBP1 to chromatin and to protect TopBP1 from proteasomal degradation. TopBP1 protein that is not bound to chromatin is ubiquitilated by HECTH9 (HUWE1) ligase. Expression of Myc leads to dissociation of TopBP1 from chromatin and reduces the amount of total TopBP1 (Herold et al., 2008). Furthermore, TopBP1 has been shown to be ubiquitilated by ubiquitin ligase EDD/hHYD (E3 identified by differential display/ human hyperplastic discs), another HECT (homologous to E6-AP C-terminus) domain E3 enzymes. The HECT E3 ubiquitin-protein ligases have been found from yeast to humans. They are characterized by the HECT domain. EDD/hHYD interacts with the minimal region of the amino acids 661 – 1080 including BRCT5 and BRCT6 of TopBP1 protein. TopBP1 was found to be usually ubiqitilated and degraded by the proteasome in intact cells. X-irradiation seems to abolish TopBP1 degradation and induce the stable complex formation of TopBP1 with other molecules in DNA double strand breaks (Honda et al., 2002; Scheffrer & Staub, 2007). Binding of the transcription factor Miz-1 and TopBP1 protein is also regulated by TopBP1 ADP-ribosylation (Table 1). ADP-ribosylation is one of the post-translational protein modifications. Polymers of ADP-ribose are formed from donor NAD+ molecules and covalently attached to glutamic acid, aspartic acid or lysine residues of a target protein. The process is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of proteins. The best known of these proteins is PARP1 which is implicated in transcription, chromatin remodeling, apoptosis and DNA repair (Sokka et al., 2010; Woodhouse & Dainov, 2008). TopBP1 and PARP-1 interact both *in vitro* and *in vivo*. The interaction depends on sixth BRCT domain of TopBP1 and on the fact that this domain is ADP-ribosylated by PARP-1. The post-translational ADP-ribosylation of TopBP1 by PARP1 may support the release of

interaction is mediated by ePHD (extended plant homeodomain) domain of SPBP and the BRCT6 domain of TopBP1 (Sjottem et al., 2007). SPBP a 220 kDa ubiquitously expressed nuclear protein is shown to intensify or repress the transcriptional activity. Originally SPBP was identified as a protein involved in transcriptional activation of matrix metalloproteinase-3 (MMP3), stromelysin-1 promoter *via* the specific sequence element SPRE (stromelysin-1 PDGF responsive element) (Rekdal et al., 2000; Sonz et al., 1995). Later SPBP was found to act as a transcriptional coactivator since it enhanced the transcriptional activity of the positive cofactor and RING finger protein SNURF/RNF4 (small nuclear RING finger protein/RING finger protein 4) and of certain transcription factors, such as Sp1 (specificity protein 1), Ets (E-twenty-six specific), Pax6 (paired box gene 6) and Jun (Lyngso et al., 2000; Rekdal et al., 2000; Sjottem et al., 2007). On the other hand, SPBP appears to act as phosphoserine-specific repressor of estrogen receptor α (ERα) (Gburick et al., 2005;

In unstressed cells TopBP1 protein associates with Miz-1 (Myc interacting zinc finger protein 1). BRCT6 and BRCT7 of TopBP1 are required and largely sufficient to mediate the interaction with Miz-1 (Fig. 2) (Herold et al., 2002, 2008; Wenzel et al., 2003). This zinc finger protein that contains an amino-terminal POZ (poxvirus and zinc finger) was initially described as a protein that interacts with C terminus of Myc oncoprotein (Courapied et al., 2010; Herold et al., 2008). Miz-1 protein activates transcription of genes encoding the cell cycle inhibitors p15INK46 and p21Cip1, leading to cell cycle arrest. Miz-1 can also repress transcription when it forms complexes with Myc and other transcription factors (Herold et al., 2002, 2008; Wenzel et al., 2003). In response to UV irradiation Miz-1 is released from an inhibitory complex formed with TopBP1 and binds to the start site of *p21Cip1* promoter. Thus the dissociation of TopBP1 from Miz-1 may facilitate the induction of *p21Cip1* (Herold et al., 2002, 2008; Wenzel et al., 2003). On the other hand, Miz-1 is required for the binding of TopBP1 to chromatin and to protect TopBP1 from proteasomal degradation. TopBP1 protein that is not bound to chromatin is ubiquitilated by HECTH9 (HUWE1) ligase. Expression of Myc leads to dissociation of TopBP1 from chromatin and reduces the amount of total TopBP1 (Herold et al., 2008). Furthermore, TopBP1 has been shown to be ubiquitilated by ubiquitin ligase EDD/hHYD (E3 identified by differential display/ human hyperplastic discs), another HECT (homologous to E6-AP C-terminus) domain E3 enzymes. The HECT E3 ubiquitin-protein ligases have been found from yeast to humans. They are characterized by the HECT domain. EDD/hHYD interacts with the minimal region of the amino acids 661 – 1080 including BRCT5 and BRCT6 of TopBP1 protein. TopBP1 was found to be usually ubiqitilated and degraded by the proteasome in intact cells. X-irradiation seems to abolish TopBP1 degradation and induce the stable complex formation of TopBP1 with other molecules in DNA double strand breaks (Honda et al., 2002; Scheffrer & Staub, 2007). Binding of the transcription factor Miz-1 and TopBP1 protein is also regulated by TopBP1 ADP-ribosylation (Table 1). ADP-ribosylation is one of the post-translational protein modifications. Polymers of ADP-ribose are formed from donor NAD+ molecules and covalently attached to glutamic acid, aspartic acid or lysine residues of a target protein. The process is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of proteins. The best known of these proteins is PARP1 which is implicated in transcription, chromatin remodeling, apoptosis and DNA repair (Sokka et al., 2010; Woodhouse & Dainov, 2008). TopBP1 and PARP-1 interact both *in vitro* and *in vivo*. The interaction depends on sixth BRCT domain of TopBP1 and on the fact that this domain is ADP-ribosylated by PARP-1. The post-translational ADP-ribosylation of TopBP1 by PARP1 may support the release of

Sjottem et al., 2007).


Miz-1 from the complex with TopBP1 (Wollmann et al., 2007; Yamane et al., 1997; Yamane & Tsuruo, 1999).

Table 1. Post-translation modifications of the human TopBP1 protein (N/D – not determined)

Apart from the mentioned above ADP-ribosylation, TopBP1 undergoes other posttranslational modifications, such as acetylation and phosphorylation (Table 1). Lysine acetylation is a reversible post-translational modification, which neutralizes the positive charge of this amino acid changing protein function. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport and actin nucleation. Acetylation of TopBP1 protein occurs at position 581 but the exact role of this modification remains to be resolved (Choudhary et al., 2010).

TopBP1 is a phosphoprotein and is phosphorylated in response to DNA damage (Makiniemi et al., 2001; Yamane et al., 2003). After DNA damage, TopBP1 protein localizes at IR-induced nuclear foci and is phosphorylated by ATM kinase (Yamane et al.,

TopBP1 in DNA Damage Response 291

with Cdc45 and this interaction inhibits transcriptional activity of TopBP1 (Schmidt et al., 2008; Sokka et al., 2010). Both proteins interact exclusively at the G1/S boundary of cell cycle. Only weak interaction could be found at the G2/M boundary (Schmidt et al., 2008).

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

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;

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

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

(Choi et al., 2009; Smits et al., 2010).

Olson et al., 2006).

2003). Human TopBP1 is phosphorylated at several S/TQ sites, which are consensus sequences of PIKK (phosphatidylinositol 3-kinase*-*related kinase) targets (Hashimoto et al., 2006; Matsuoka et al., 2007). However, the phosphorylation of TopBP1 protein occurs mostly on serine and to a lesser extent on threonine (Makiniemi et al., 2001). TopBP1 protein is also phosphorylated by Akt *in vitro* and *in vivo* on Ser1159. Phosphorylation by Akt kinase induces oligomerization of TopBP1 through its seventh and eighth BRCT domains. The Akt-dependent oligomerization is crucial for TopBP1 to interact with E2F1 and repress its activity. TopBP1 phosphorylation by Akt is also required for interaction between TopBP1 and Miz-1 or HPV16 E2 and repression of Miz-1 transcriptional activity, suggesting a general role for TopBP1 oligomerization in the control of transcription factors (Liu et al., 2006a).

The other TopBP1 interacting proteins are PML (promyelocytic leukemia protein), TICRR (TopBP1-interacting, checkpoint and replication regulator) and p53. PML is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation and genome stability. PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. PML colocalizes and associates *in vivo* with TopBP1 in response to ionizing radiation and both proteins colocalize with Rad50, BRCA1, ATM, Rad9 and BLM. PML plays a role in regulation of TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage (Xu et al., 2003). TICRR is required to prevent mitotic entry after treatment with ionizing radiation. TICRR deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of TICRR impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. TICRR associates with TopBP1 *in vivo* and this interaction requires the two N-terminal BRCT domains. Sansam et al. (2010) showed that interaction between TICRR and TopBP1 is essential for replication preinitiation complex. TopBP1 is also involved in regulation of p53 activity. The regulation is mediated by an interaction between the seventh and eighth BRCT domains of TopBP1 and the DNA binding domain of p53, leading to inhibition of p53 promoter binding activity. Thus, TopBP1 may inhibit expression of several canonic p53 target genes including GADD45 (growth arrest and DNA damage protein 45), p21Cip1, PUMA (p53 upregulated modulator of apoptosis), NOXA, BAX (Bcl-2 associated X protein), IGFBP3 (insulin-like growth factor binding protein 3). The repression of p53 proapoptotic genes such as NOXA, PUMA and BAX suggests that TopBP1 can inhibit p53-mediated apoptosis during DNA damage. Deregulation of this control may have pathological consequences (Liu et al., 2009).

TopBP1 also plays a role in DNA replication and S phase progression. Expression of TopBP1 mRNA and protein is induced concomitantly with S phase entry (Makiniemi et al., 2001). Neutralizing TopBP1 with a polyclonal antiserum raised against the sixth BRCT domain inhibits replicative DNA synthesis in HeLa cell nuclei *in vitro*. This may indicate that the sixth BRCT domain is critical for replication activity, possibly *via* interaction with crucial replication factors (Makiniemi et al., 2001; Schmidt et al., 2008). The physical interaction between TopBP1 and polymerase ε also implies an involvement of TopBP1 in replication (Makiniemi et al., 2001). The loading of Cdc45 (cell division cycle 45) onto chromatin is critical for loading various replication proteins, including DNA polymerase α, DNA polymerase ε, RPA (replication protein A) and PCNA. Human TopBP1 recruits Cdc45 to origins of DNA replication and is required for the formation of the initiation complex of replication in human cells. The first, second and sixth BRCT domains of TopBP1 interact

2003). Human TopBP1 is phosphorylated at several S/TQ sites, which are consensus sequences of PIKK (phosphatidylinositol 3-kinase*-*related kinase) targets (Hashimoto et al., 2006; Matsuoka et al., 2007). However, the phosphorylation of TopBP1 protein occurs mostly on serine and to a lesser extent on threonine (Makiniemi et al., 2001). TopBP1 protein is also phosphorylated by Akt *in vitro* and *in vivo* on Ser1159. Phosphorylation by Akt kinase induces oligomerization of TopBP1 through its seventh and eighth BRCT domains. The Akt-dependent oligomerization is crucial for TopBP1 to interact with E2F1 and repress its activity. TopBP1 phosphorylation by Akt is also required for interaction between TopBP1 and Miz-1 or HPV16 E2 and repression of Miz-1 transcriptional activity, suggesting a general role for TopBP1 oligomerization in the control of transcription

The other TopBP1 interacting proteins are PML (promyelocytic leukemia protein), TICRR (TopBP1-interacting, checkpoint and replication regulator) and p53. PML is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation and genome stability. PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. PML colocalizes and associates *in vivo* with TopBP1 in response to ionizing radiation and both proteins colocalize with Rad50, BRCA1, ATM, Rad9 and BLM. PML plays a role in regulation of TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage (Xu et al., 2003). TICRR is required to prevent mitotic entry after treatment with ionizing radiation. TICRR deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of TICRR impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. TICRR associates with TopBP1 *in vivo* and this interaction requires the two N-terminal BRCT domains. Sansam et al. (2010) showed that interaction between TICRR and TopBP1 is essential for replication preinitiation complex. TopBP1 is also involved in regulation of p53 activity. The regulation is mediated by an interaction between the seventh and eighth BRCT domains of TopBP1 and the DNA binding domain of p53, leading to inhibition of p53 promoter binding activity. Thus, TopBP1 may inhibit expression of several canonic p53 target genes including GADD45 (growth arrest and DNA damage protein 45), p21Cip1, PUMA (p53 upregulated modulator of apoptosis), NOXA, BAX (Bcl-2 associated X protein), IGFBP3 (insulin-like growth factor binding protein 3). The repression of p53 proapoptotic genes such as NOXA, PUMA and BAX suggests that TopBP1 can inhibit p53-mediated apoptosis during DNA damage. Deregulation of this control may

TopBP1 also plays a role in DNA replication and S phase progression. Expression of TopBP1 mRNA and protein is induced concomitantly with S phase entry (Makiniemi et al., 2001). Neutralizing TopBP1 with a polyclonal antiserum raised against the sixth BRCT domain inhibits replicative DNA synthesis in HeLa cell nuclei *in vitro*. This may indicate that the sixth BRCT domain is critical for replication activity, possibly *via* interaction with crucial replication factors (Makiniemi et al., 2001; Schmidt et al., 2008). The physical interaction between TopBP1 and polymerase ε also implies an involvement of TopBP1 in replication (Makiniemi et al., 2001). The loading of Cdc45 (cell division cycle 45) onto chromatin is critical for loading various replication proteins, including DNA polymerase α, DNA polymerase ε, RPA (replication protein A) and PCNA. Human TopBP1 recruits Cdc45 to origins of DNA replication and is required for the formation of the initiation complex of replication in human cells. The first, second and sixth BRCT domains of TopBP1 interact

factors (Liu et al., 2006a).

have pathological consequences (Liu et al., 2009).

with Cdc45 and this interaction inhibits transcriptional activity of TopBP1 (Schmidt et al., 2008; Sokka et al., 2010). Both proteins interact exclusively at the G1/S boundary of cell cycle. Only weak interaction could be found at the G2/M boundary (Schmidt et al., 2008).
