**3. Checkpoint signaling cascade**

Proteins of checkpoint signaling pathways are classified as sensors, transducers and effectors (Fig. 1). Following DNA damage, sensor multiprotein complexes, e.g. MRN (MRE11-Rad50-NBS1) or 9-1-1 (Rad9-Rad1-Hus1) recognize damage and recruit proximal transducers, i.e. ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases to lesions where they are initially activated. ATM and ATR transduce signals to distal transducer, i.e. checkpoint kinases Chk1 and Chk2 (Dai & Grant, 2010; Niida & Nakanishi, 2006). Chk1 and Chk2 kinases, distal transducers, transfer the signal of DNA damage to effectors, such as Cdks (cyclin-dependent kinases), Cdc25 (cell division cycle 25) and p53 (Dai & Grant, 2010; Houtgraaf et al., 2006; Nakanishi, 2009; Nakanishi et al., 2009). The key difference between ATM and ATR is the signal that activates them. ATM is

TopBP1 in DNA Damage Response 285

In addition to damage sensors and signal transducers, many other proteins called mediators are involved in DNA damage response. Mediators are mostly cell cycle specific proteins associated with damage sensors and signal transducers at particular phases of the cell cycle and, as a consequence, help provide signal transduction specificity. ATM and ATR phosphorylate most of these mediators. Well-known examples of mediators are 53BP1 (p53 binding protein 1), MDC1 (mediator of DNA damage checkpoint 1), BRCA1 (breast cancer 1), SMC1 (structural maintenance of chromosomes 1), FANCD2 (Fanconi anemia, complementation group D2), Claspin, Timeless, Tipin and histone H2AX (Dai & Grant, 2010; Houtgraaf et al., 2006; Yang et al., 2010). This group of regulators involves also TopBP1 protein (topoisomerase IIβ binding protein 1) (Cimprich & Cortez, 2008). Certain molecules may have multiple functions in this signal transduction pathway. For example ATM and ATR can simultaneously act as a sensor and a transducer. Consequently, signal transduction in DNA damage response is not one-dimensional but a complex network of interacting

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,

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

*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).

molecules (Poehlmann & Roessner, 2010).

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

2003; Taricani & Wand, 2006; van Hatten et al., 2002).

primary cells (Bang et al., 2011; Jeon et al., 2011).

activated exclusively by DSBs, which can arise from endogenous (ROS, eroded telomeres, intermediates of immune and meiotic recombination) or exogenous (IR, genotoxic drugs) sources (Lopez-Contreras & Fernandez-Capetillo, 2010). In contrast, ATR responds to many types of DNA damage and replication stress including breaks, crosslinks and base adducts. ATR senses abnormally long stretches of single strand DNA that arise from the functional uncoupling of helicase and polymerase activities at replication forks or from the processing of DNA lesions such as the resection of DSBs (Mordes & Cortez, 2008). ATR but not ATM is essential for viability. The early embryonic death in ATR knockout mice shows that ATR is essential for cell growth and differentiation at an early stage of development (Smits et al., 2010). In addition, disruption of ATR in mouse or human cells results in cell cycle arrest or death, even without exogenous DNA damage (Cortez et al., 2001; Smits et al., 2010). Although complete inactivation of ATR is lethal, a hypomorphic mutation was found in humans suffering from the rare autosomal recessive disorder, Seckel syndrome, characterized by growth retardation and microcephaly. In homozygosity, that mutation affects ATR splicing which results in the reduction of ATR protein levels to almost undetectable, yet the remaining protein is sufficient for viability (Kerzendorfer & O'Driscoll, 2009; O'Driscoll et al., 2004; Smits et al., 2010).

Fig. 1. Signal transduction of DNA damage response (DDR)

activated exclusively by DSBs, which can arise from endogenous (ROS, eroded telomeres, intermediates of immune and meiotic recombination) or exogenous (IR, genotoxic drugs) sources (Lopez-Contreras & Fernandez-Capetillo, 2010). In contrast, ATR responds to many types of DNA damage and replication stress including breaks, crosslinks and base adducts. ATR senses abnormally long stretches of single strand DNA that arise from the functional uncoupling of helicase and polymerase activities at replication forks or from the processing of DNA lesions such as the resection of DSBs (Mordes & Cortez, 2008). ATR but not ATM is essential for viability. The early embryonic death in ATR knockout mice shows that ATR is essential for cell growth and differentiation at an early stage of development (Smits et al., 2010). In addition, disruption of ATR in mouse or human cells results in cell cycle arrest or death, even without exogenous DNA damage (Cortez et al., 2001; Smits et al., 2010). Although complete inactivation of ATR is lethal, a hypomorphic mutation was found in humans suffering from the rare autosomal recessive disorder, Seckel syndrome, characterized by growth retardation and microcephaly. In homozygosity, that mutation affects ATR splicing which results in the reduction of ATR protein levels to almost undetectable, yet the remaining protein is sufficient for viability (Kerzendorfer & O'Driscoll,

2009; O'Driscoll et al., 2004; Smits et al., 2010).

Fig. 1. Signal transduction of DNA damage response (DDR)

In addition to damage sensors and signal transducers, many other proteins called mediators are involved in DNA damage response. Mediators are mostly cell cycle specific proteins associated with damage sensors and signal transducers at particular phases of the cell cycle and, as a consequence, help provide signal transduction specificity. ATM and ATR phosphorylate most of these mediators. Well-known examples of mediators are 53BP1 (p53 binding protein 1), MDC1 (mediator of DNA damage checkpoint 1), BRCA1 (breast cancer 1), SMC1 (structural maintenance of chromosomes 1), FANCD2 (Fanconi anemia, complementation group D2), Claspin, Timeless, Tipin and histone H2AX (Dai & Grant, 2010; Houtgraaf et al., 2006; Yang et al., 2010). This group of regulators involves also TopBP1 protein (topoisomerase IIβ binding protein 1) (Cimprich & Cortez, 2008). Certain molecules may have multiple functions in this signal transduction pathway. For example ATM and ATR can simultaneously act as a sensor and a transducer. Consequently, signal transduction in DNA damage response is not one-dimensional but a complex network of interacting molecules (Poehlmann & Roessner, 2010).
