**4. Structure of TopBP1 and its similarity to BRCA1**

Topoisomerase IIβ binding protein 1 (TopBP1) has been identified as a protein interacting with topoisomerase IIβ in a yeast two-hybrid screen (Morishima et al., 2007; Yamane et al., 1997). Interaction with topoisomerase IIβ is mediated by carboxyl-terminal region (aa 862- 1522) of TopBP1 *in vitro* (Honda et al., 2002; Yamane et al., 1997). TopBP1 shares sequence and structural homologies with *Saccharomyces cerevisiae* Dpb11, *Schizosaccharomyces pombe* Cut5/Rad4, *Drosophila melanogaster* Mus101 and *Xenopus levis* Xmus101 (Araki et al., 1995; Garcia et al., 2005; Morishima et al., 2007; Ogiwara et al., 2006; Parrilla-Castellar & Karnitz, 2003; Taricani & Wand, 2006; van Hatten et al., 2002).

TopBP1 protein seems to be essential for maintenance of chromosomal integrity and cell proliferation. This protein appeared to be involved in DNA damage response, DNA replication checkpoint, chromosome replication and regulation of transcription (Bang et al., 2011; Garcia et al., 2005; Jeon et al., 2011). TopBP1 knockout mouse exhibits early embryonic lethality at the peri-implantation stage and TopBP1 deficiency induces cellular senescence in primary cells (Bang et al., 2011; Jeon et al., 2011).

*TopBP1* gene comprising 28 exons is located on chromosome 3q22.1 and encodes a 1522 amino acid protein (180 kDa) (Karppinen et al., 2006; Xu & Leffak, 2010; Yan & Michael, 2009a,b). The structure of protein is characterized by the presence of interspersed throughout the whole molecule eight copies of the BRCT domain (C-terminal domain of BRCA1), originally identified as a tandemly repeated sequence motif in carboxyl-terminal region of BRCA1 (Fig. 2) (Glover, 2006; Lelung et al., 2010; Wright et al., 2006; Yamane et al., 1997; Yamane & Tsuruo, 1999). BRCT domains, about 90 amino acids in length, are hydrophobic and are involved in an interaction with other proteins and phosphorylated peptides, as well as in an interaction with single- and double-stranded DNA (Glover, 2006; Rodriquez et al., 2003; Wright et al., 2006). A sequence analysis has shown that BRCT repeats are present in a large family of proteins that are implicated in the cellular response to DNA damage. Next to BRCA1 and TopBP1, members of this family include several proteins that are directly linked to DNA repair and cell cycle checkpoints, such as XRCC1 (X-ray cross complementing protein 1), DNA ligase III and IV, MDC1, BARD1 (BRCA1 associated RING domain protein 1), Rad9, MCPH1 (microcephalin 1) (Glover, 2006; Glover et al., 2004; Hou et al., 2010; Yamane et al., 2002; Yamane & Tsuruo, 1999; Yang et al., 2008).

TopBP1 in DNA Damage Response 287

activation domain. Additionally, another repressor domain exists on the C terminus of the activation domain, which requires amino acids 586 – 675. TopBP1 protein exerts its function in the nucleus and the carboxyl-terminal region of TopBP1 contains two putative nuclear localization signals (Going et al., 2007; Liu et al., 2003; Sokka et al., 2010). Liu et al. (2003) showed that deletion of the BRCT7-8 and NLS region of TopBP1 induces cytoplasmic localization of the protein. Aberrant expression and intracellular localization of TopBP1 is

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

observed immunohistochemically in breast cancer (Going et al., 2007).

regulation to prevent apoptosis during DNA replication (Liu et al., 2003).

E2 to activate transcription and replication (Fig. 2) (Boner et al., 2002).

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

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

**5. TopBP1 as multifunctional protein** 

Fig. 2. TopBP1 functional domains and sites of interacting proteins

The carboxyl-terminal region of TopBP1 containing two BRCT domains shows considerable similarity to the corresponding part of BRCA1 (Going et al., 2007; Karppinen et al., 2006; Makiniemi et al., 2001; Morris et al., 2009; Yamane et al., 1997, 2003). Apart from structural similarity TopBP1 shares many other common features with BRCA1. The expression of both proteins is the highest in S phase cells. TopBP1 and BRCA1 are phosphorylated by ATM in response to DNA damage and DNA replication stress and they both colocalize with PCNA (proliferating cell nuclear antigen) at stalled replication forks (Makiniemi et al., 2001; Yamane et al., 2003). The localization patterns of TopBP1 and BRCA1 have similarities also during late mitosis, as well as in meiotic prophase I (Karppinen et al., 2006; Reini et al., 2004). Furthermore, the two proteins have been shown to possess overlapping functions in G2/M checkpoint regulation (Karppinen et al., 2006). Yamane et al. (2003) demonstrated that a BRCA1-mutant or a TopBP1-reduced background results in only partial abrogation at G2/M checkpoint, whereas the combined TopBP1-reduced and BRCA-mutant background result in the nearly complete abrogation. In response to ionizing radiation TopBP1 and BRAC1 colocalize with Rad50, ATM, Rad9, BLM (Bloom syndrome protein), PCNA, NBS1 (Nijmegen breakage syndrome 1) and γH2AX in IR-induced nuclear foci (Germann et al., 2010; Xu et al., 2003).

TopBP1 protein possesses transcriptional-activation domain and two surrounding repressor domains and can play a role in regulating transcription directly (Fig. 2). A transcriptionalactivation domain is located between amino acids 460 - 591 and partly contains BRCT4 domain. This region essential for transactivation is rich in hydrophobic amino acids interspersed with acidic residues, typical of identified transcriptional domains. On aminoterminal side of the transcriptional activation domain, Wright et al., (2006) identified a repressor domain involving BRCT2 that is able to repress the TopBP1 transcriptional

Fig. 2. TopBP1 functional domains and sites of interacting proteins

2010; Xu et al., 2003).

The carboxyl-terminal region of TopBP1 containing two BRCT domains shows considerable similarity to the corresponding part of BRCA1 (Going et al., 2007; Karppinen et al., 2006; Makiniemi et al., 2001; Morris et al., 2009; Yamane et al., 1997, 2003). Apart from structural similarity TopBP1 shares many other common features with BRCA1. The expression of both proteins is the highest in S phase cells. TopBP1 and BRCA1 are phosphorylated by ATM in response to DNA damage and DNA replication stress and they both colocalize with PCNA (proliferating cell nuclear antigen) at stalled replication forks (Makiniemi et al., 2001; Yamane et al., 2003). The localization patterns of TopBP1 and BRCA1 have similarities also during late mitosis, as well as in meiotic prophase I (Karppinen et al., 2006; Reini et al., 2004). Furthermore, the two proteins have been shown to possess overlapping functions in G2/M checkpoint regulation (Karppinen et al., 2006). Yamane et al. (2003) demonstrated that a BRCA1-mutant or a TopBP1-reduced background results in only partial abrogation at G2/M checkpoint, whereas the combined TopBP1-reduced and BRCA-mutant background result in the nearly complete abrogation. In response to ionizing radiation TopBP1 and BRAC1 colocalize with Rad50, ATM, Rad9, BLM (Bloom syndrome protein), PCNA, NBS1 (Nijmegen breakage syndrome 1) and γH2AX in IR-induced nuclear foci (Germann et al.,

TopBP1 protein possesses transcriptional-activation domain and two surrounding repressor domains and can play a role in regulating transcription directly (Fig. 2). A transcriptionalactivation domain is located between amino acids 460 - 591 and partly contains BRCT4 domain. This region essential for transactivation is rich in hydrophobic amino acids interspersed with acidic residues, typical of identified transcriptional domains. On aminoterminal side of the transcriptional activation domain, Wright et al., (2006) identified a repressor domain involving BRCT2 that is able to repress the TopBP1 transcriptional activation domain. Additionally, another repressor domain exists on the C terminus of the activation domain, which requires amino acids 586 – 675. TopBP1 protein exerts its function in the nucleus and the carboxyl-terminal region of TopBP1 contains two putative nuclear localization signals (Going et al., 2007; Liu et al., 2003; Sokka et al., 2010). Liu et al. (2003) showed that deletion of the BRCT7-8 and NLS region of TopBP1 induces cytoplasmic localization of the protein. Aberrant expression and intracellular localization of TopBP1 is observed immunohistochemically in breast cancer (Going et al., 2007).
