**TopBP1 in DNA Damage Response**

Ewa Forma, Magdalena Brys and Wanda M. Krajewska *Department of Cytobiochemistry, University of Lodz Poland* 

#### **1. Introduction**

280 DNA Repair

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Jensen, S.; Bartek, J. & Shiloh, Y. (2006). Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent DNA, the genetic material of cells, is constantly exposed to a range of endogenous and environmental damaging agents (Jungmichel & Stucki, 2010). DNA molecule is the target of endogenous cellular metabolites such as reactive oxygen species (ROS) (Ciccia & Elledge, 2010; Poehlmann & Roessner, 2010). ROS may cause different alterations in a genome, e.g. simple DNA mutations, DNA single and double strand breaks (SSBs and DSBs, respectively), or more complex changes, including deletions, translocations and fusions (Poehlmann & Roessner, 2010). Alterations may be generated spontaneously due to dNTP misincorporation during DNA replication, interconversion between DNA bases caused by deamination, loss of DNA bases following DNA depurination or depyrimidination and modification of DNA bases by alkylation. Hydrolytic deamination (loss of an amino group) can directly convert one base to another. For example, deamination of cytosine results in uracil and with much lower frequency converts adenine to hypoxanthine. In depurination or depyrimidination, purine or pyrimidine bases are completely removed, leaving deoxyribose sugar depurinated or depyrimidinated that may cause breakage in the DNA backbone (Ciccia & Elledge, 2010; Rastogi et al., 2010). Altogether, it has been estimated that every cell could experience up to 105 spontaneous DNA lesions per day (Ciccia & Elledge, 2010). Environmental DNA damage can be produced by physical or chemical sources, such as ionizing radiation (IR), ultraviolet (UV) light from sunlight and organic and inorganic chemical substances (Muniandy et al., 2010; Rastogi et al., 2010; Su et al., 2010). Exposure to ionizing radiation from, e.g. cosmic radiation and medical treatments employing X-rays or radiotherapy inflicts DNA single and double strand breaks, oxidation of DNA bases and DNA-protein crosslinks in the genomic DNA (Ciccia & Elledge, 2010; Su et al., 2010). Ionizing radiation provokes DNA damage directly by energy deposit on the DNA double helix and indirectly by reactive oxygen/nitrogen species (ROS/RNS) (Corre et al., 2010). Ultraviolet radiation (mainly UV-B) is a powerful agent that may lead to the formation of three major classes of DNA lesions, such as cyclobutane pyrimidine dimmers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs) and their Devar isomers (Rastogi et al., 2010). Cells may become transiently exposed to external sources of DNA damage, such as cigarette smoke or various toxic chemical compounds (Jungmichel & Stucki, 2010). Many antineoplastic drugs currently used in cancer treatment express their cytotoxic effects through their ability to directly or indirectly damage DNA and thus resulting in cell death. Major types of DNA damage induced by anticancer treatment include single and double strand breaks, interstrand, intrastrand and DNA-protein crosslinks, as well as interference

TopBP1 in DNA Damage Response 283

Together DNA repair mechanisms and DNA damage signaling system form a molecular

To maintain genomic integrity and faithful transmission of fully replicated and undamaged DNA during cell division, eukaryotic organisms evolved a complex DNA surveillance program (Reihardt & Yaffe, 2009). Apart from DNA repair mechanisms mentioned above, DNA damage response represents a complex network of multiple signaling pathways involving cell cycle checkpoints, transcriptional regulation, chromatin remodeling and apoptosis (Dai & Grant, 2010; Danielsen et al., 2009). In response to DNA damage, eukaryotic cells activate a complex protein kinase-based signaling network to arrest progression through the cell cycle. Activation of signaling cascade recruits repair machinery to the site of DNA damage, provides time for repair or if the genotoxic insult exceeds repair capacity, additional signaling pathways leading to cell death, presumably *via* apoptosis, are activated (Reinhardt et al., 2010; Reinhardt & Yaffe, 2009). When DNA damage occurs, distinct, albeit overlapping and cooperating checkpoint pathways are activated, which block S phase entry (the G1/S phase checkpoint), delay S phase progression (the S phase checkpoints) or prevent mitotic entry (the G2/M phase checkpoint). The primary G1/S cell cycle checkpoint controls the commitment of eukaryotic cells to transition through G1 phase and enter DNA synthesis phase. In G1 phase, cells have to make a decision between continuing proliferation or exiting the cell cycle to become quiescent differentiated, senescent or apoptotic (Dijkstra et al., 2009). The S phase checkpoints are activated when DNA damage occurs during DNA synthesis, or when DNA replication intermediates accumulate. Depending on the type and magnitude of damage, cells activate one of three distinct S phase checkpoint pathways: an intra-S phase checkpoint induced by double strand breaks, a replication checkpoint by the stalled replication fork and the S/M checkpoint blocking premature mitosis. The S/M checkpoint differs from the well-defined G2/M checkpoint. The S/M checkpoint is ATM-independent, it is measurable only several hours after DNA damage and is initiated in cells that were in S phase at the time of insult (Hurley & Bunz, 2009; Rodriguez-Bravo et al., 2007). When cells encounter DNA damage in G2, the G2/M checkpoint stops the cell cycle to prevent the cell from entering mitosis. Defects in cell cycle arrest at the respective checkpoint are associated with genome

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

shield against genomic instability.

**2. DNA damage checkpoints** 

instability and oncogenesis (Houtgraaf et al., 2006).

**3. Checkpoint signaling cascade** 

with nucleotide metabolism and DNA synthesis (Pallis & Karamouzis, 2010). Alkylating agents, such as methyl methanesulphonate (MMS), tenozalamide, streptozotocin, procarbazine, dacarbazine, ethylnitrosourea, diethylnitrosamine and nitrosoureas attach alkyl groups to DNA bases, while crosslinking agents such as mitomycin (MMC), cisplatin, psoralen and nitrogen mustard induce covalent links between bases of the same DNA strand (intrastrand crosslinks) or of different DNA strands (interstrands crosslinks) (Ciccia & Elledge, 2010; Muniandy et al., 2010; Pallis & Karamouzis, 2010). Other chemical agents, such as topoisomerase inhibitors induce the formation of single or double strand breaks by trapping topoisomerase-DNA covalent complexes (Ciccia & Elledge, 2010). Camptothecin and novel noncamptothecins in clinical development target eukaryotic IB type topoisomerase (Topo I), whereas human IIA type topoisomerases (Topo IIα and Topo IIβ) are the targets of widely used anticancer agents, such as etoposide, anthracyclines (doxorubicin, daunorubicin) and mitoxantrone (Pommier et al., 2010).

The biochemical consequences of DNA lesions are diverse and range from obstruction of fundamental cellular pathways like transcription and replication to fixation of mutations. Cellular misfunctioning, cell death, aging and cancer are the phenotypical consequences of DNA damage accumulation in the genome. To counteract DNA damage, repair mechanisms specific for many types of lesions have evolved. Mispaired DNA bases are replaced with correct bases by mismatch repair (MMR) (Ciccia & Elledge, 2010). The bases excision repair (BER) exerts its biological role by removing bases that have been damaged by alkylation, oxidation, ring saturation, as well as a short strand that contains the damaged bases. BER also plays an important role in the repair of DNA single strand breaks generated spontaneously or induced by exogenous DNA-damaging factors such as cytotoxic anticancer agents (Pallis & Karamouzis, 2010). DNA single strand breaks may be also repaired by single strand break repair (SSBR) (Ciccia & Elledge, 2010). Nucleotide excision repair (NER) is a highly conserved pathway that repairs DNA damage caused by UV radiation, mutagenic chemicals or chemotherapeutic drugs that form bulky DNA adducts (Pallis & Karamouzis, 2010). The most toxic lesions in DNA are double strand breaks where the phosphate backbones of the two complementary DNA strands are broken simultaneously (Hiom, 2010). Double strand breaks are repaired by two major repair pathways depending on the context of DNA damage, i.e. homologous recombination (HR) and nonhomologous end-joining (NHEJ) (Hiom, 2010; Pallis & Karamouzis, 2010). While NHEJ promotes potential inaccurate relegation of double strand breaks, HR precisely restores genomic sequence of the broken DNA ends by using sister chromatids as template for repair (Ciccia & Elledge, 2010). Additionally, some specialized polymerases can temporarily take over lesion-arrested DNA polymerases during S phase, in a mutagenic mechanism called translesion synthesis (TLS). Such polymerases only work if a more reliable system, such as homologous recombination, cannot avoid stumbled DNA replication (Essers et al., 2006).

DNA repair is carried out by the plethora of enzymatic activities that chemically modify DNA to repair DNA damage, including nucleases, helicases, polymerases, topoisomerases, recombinases, ligases, glycosylases, demethylases, kinases and phosphatases. These repair tools must be precisely regulated, because each in its own right can wreak havoc on the integrity of DNA if misused or allowed to gain access to DNA at the inappropriate time or place (Ciccia & Elledge, 2010). The DNA repair mechanisms function in conjunction with an intricate machinery of damage sensors, responsible of a series of phosphorylations and chromatin modifications that signal to the rest of the cell the presence of lesions on DNA.

with nucleotide metabolism and DNA synthesis (Pallis & Karamouzis, 2010). Alkylating agents, such as methyl methanesulphonate (MMS), tenozalamide, streptozotocin, procarbazine, dacarbazine, ethylnitrosourea, diethylnitrosamine and nitrosoureas attach alkyl groups to DNA bases, while crosslinking agents such as mitomycin (MMC), cisplatin, psoralen and nitrogen mustard induce covalent links between bases of the same DNA strand (intrastrand crosslinks) or of different DNA strands (interstrands crosslinks) (Ciccia & Elledge, 2010; Muniandy et al., 2010; Pallis & Karamouzis, 2010). Other chemical agents, such as topoisomerase inhibitors induce the formation of single or double strand breaks by trapping topoisomerase-DNA covalent complexes (Ciccia & Elledge, 2010). Camptothecin and novel noncamptothecins in clinical development target eukaryotic IB type topoisomerase (Topo I), whereas human IIA type topoisomerases (Topo IIα and Topo IIβ) are the targets of widely used anticancer agents, such as etoposide, anthracyclines

The biochemical consequences of DNA lesions are diverse and range from obstruction of fundamental cellular pathways like transcription and replication to fixation of mutations. Cellular misfunctioning, cell death, aging and cancer are the phenotypical consequences of DNA damage accumulation in the genome. To counteract DNA damage, repair mechanisms specific for many types of lesions have evolved. Mispaired DNA bases are replaced with correct bases by mismatch repair (MMR) (Ciccia & Elledge, 2010). The bases excision repair (BER) exerts its biological role by removing bases that have been damaged by alkylation, oxidation, ring saturation, as well as a short strand that contains the damaged bases. BER also plays an important role in the repair of DNA single strand breaks generated spontaneously or induced by exogenous DNA-damaging factors such as cytotoxic anticancer agents (Pallis & Karamouzis, 2010). DNA single strand breaks may be also repaired by single strand break repair (SSBR) (Ciccia & Elledge, 2010). Nucleotide excision repair (NER) is a highly conserved pathway that repairs DNA damage caused by UV radiation, mutagenic chemicals or chemotherapeutic drugs that form bulky DNA adducts (Pallis & Karamouzis, 2010). The most toxic lesions in DNA are double strand breaks where the phosphate backbones of the two complementary DNA strands are broken simultaneously (Hiom, 2010). Double strand breaks are repaired by two major repair pathways depending on the context of DNA damage, i.e. homologous recombination (HR) and nonhomologous end-joining (NHEJ) (Hiom, 2010; Pallis & Karamouzis, 2010). While NHEJ promotes potential inaccurate relegation of double strand breaks, HR precisely restores genomic sequence of the broken DNA ends by using sister chromatids as template for repair (Ciccia & Elledge, 2010). Additionally, some specialized polymerases can temporarily take over lesion-arrested DNA polymerases during S phase, in a mutagenic mechanism called translesion synthesis (TLS). Such polymerases only work if a more reliable system, such as homologous recombination, cannot avoid stumbled DNA

DNA repair is carried out by the plethora of enzymatic activities that chemically modify DNA to repair DNA damage, including nucleases, helicases, polymerases, topoisomerases, recombinases, ligases, glycosylases, demethylases, kinases and phosphatases. These repair tools must be precisely regulated, because each in its own right can wreak havoc on the integrity of DNA if misused or allowed to gain access to DNA at the inappropriate time or place (Ciccia & Elledge, 2010). The DNA repair mechanisms function in conjunction with an intricate machinery of damage sensors, responsible of a series of phosphorylations and chromatin modifications that signal to the rest of the cell the presence of lesions on DNA.

(doxorubicin, daunorubicin) and mitoxantrone (Pommier et al., 2010).

replication (Essers et al., 2006).

Together DNA repair mechanisms and DNA damage signaling system form a molecular shield against genomic instability.
