**1. Introduction**

160 DNA Repair

Yuan, Z.M., Huang, Y., Ishiko, T., Nakada, S., Utsugisawa, T., Kharbanda, S., Wang, R.,

function by c-Abl in response to DNA damage. J Biol Chem *273*, 3799-3802. Zhong, Q., Chen, C.F., Li, S., Chen, Y., Wang, C.C., Xiao, J., Chen, P.L., Sharp, Z.D., & Lee,

DNA damage response. Science *285*, 747-750.

Sung, P., Shinohara, A., Weichselbaum, R., & Kufe, D. (1998). Regulation of Rad51

W.H. (1999). Association of BRCA1 with the hRad50-hMre11-p95 complex and the

Cells are continually exposed to genotoxic stresses. Upon DNA damage, the cell activates a coordinated and complex series of responses (Levitt and Hickson, 2002). Multiple factors are implicated in each of these responses. Recently, it has become apparent that various transcription factors play important roles in cellular responses to genotoxic stress. In particular, E2F transcription factors are key for the activation of genes involved in these processes.

E2F family comprises two subfamilies, termed E2F and DP, and includes orthologs expressed across many species, from plants to higher vertebrates (McClellan and Slack, 2007). In mammals, multiple E2F (E2F-1 through -8) and DP (DP-1 through -4) genes have been identified. E2F-1, -2 and -3 are associated with DNA synthesis and cell cycle progression, and function as heterodimers with a DP member (McClellan and Slack, 2007). E2F-4 and -5 also require association with a DP protein, but often function to halt cell cycle progression associated with terminal differentiation or reversible entry into quiescence (McClellan and Slack, 2007). E2F-1 through -5 can mediate transcriptional activation when found as "free" E2F/DP dimers, but can also act as transcriptional repressors if they are associated with a member of the retinoblastoma (pRb) family of proteins (Hallstrom and Nevins, 2009). In contrast, E2F-6 lacks both transcriptional activation and pRb-binding domains, and functions as a constitutive transcriptional repressor. The most divergent members of the E2F family are E2F-7 and -8, which bind neither DP nor pRb-family proteins, and also function as transcriptional repressors to mediate cell cycle arrest (Lammens et al., 2009). To regulate gene expression, E2F factors bind GC-rich elements on proximal promoters, which can conform to either a consensus 5'-TTTC[CG]CGC-3' element, or to non-consensus sequences (Judah et al., 2010; Rabinovich et al., 2008). Considerable efforts have been directed to investigate whether different E2F proteins exhibit target selectivity. Genome-wide screens for E2F targets have revealed considerable overlap in the ability of individual E2F proteins to regulate their targets, although a few promoters activated by specific E2F forms have been identified (Cao et al., 2011).

Post-Transcriptional Regulation of E2F Transcription Factors: Fine-Tuning

**2.1 Identification of E2F targets involved in DNA damage repair** 

carcinogenesis, but also the potential impact of various therapies.

actively transcribed regions (Nouspikel, 2009).

**2.2 Role of E2F-1 in responses to DNA damage induced by UV radiation** 

UV radiation induces severe DNA damage, which is the principal cause of skin carcinogenesis in humans (Brash et al., 1996). UV-B radiation induces formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6- 4PP), which would result in loss of DNA integrity and genetic instability if left unrepaired. This type of damage to DNA triggers activation of the nucleotide-excision repair pathway, and can occur *via* one or more streams. Such DNA repair streams include (i) global genome repair (GGR), which repairs damage from the entire genome, (ii) transcription-coupled repair (TCR), which generally repairs damage on actively transcribed DNA strands & (iii) transcription domain-associated repair (DAR), which deals with repairing both strands of

Normal responses of the epidermis to UV damage are critically dependent on E2F-1 expression. Indeed, increased levels of epidermal apoptosis upon UV-B irradiation have been reported in E2F-1-null mouse epidermis, whereas repair of UV-B-induced DNA photoproducts is more efficient in keratinocytes that overexpress E2F-1 (Berton et al., 2005). UV-induced DNA damage results in stabilization of E2F-1 protein, which stimulates nucleotide excision repair (Berton et al., 2005; Pediconi et al., 2003; Wikonkal et al., 2003). The mechanisms involved include phosphorylation of E2F-1 on Ser31 by ATR and/or ATM kinases (Lin et al., 2001). This modification facilitates E2F-1 recruitment to sites of doublestrand breaks or UV-induced DNA damage. Under these conditions, E2F-1 interacts with two key proteins involved in DNA repair: TopBP1 and GCN5 histone acetyltransferase (Guo et al., 2010a; Guo et al., 2010b). Formation of these E2F-1 complexes is necessary for efficient recruitment of factors involved in nucleotide excision repair. Importantly, the association of E2F-1 with TopBP1 and GCN5 occurs at the expense of the E2F-1-induced expression of proapoptotic p73, thus ensuring that DNA repair, rather than apoptosis, takes place (Berton et al., 2005; Pediconi et al., 2003; Wikonkal et al., 2003). In mouse embryo fibroblasts, UV-C irradiation results in the formation of both CPD and 6-4PP. In these cells, nucleotide excision repair is activated through pathways that involve activation of xeroderma pigmentosum

DNA Repair, Cell Cycle Progression and Survival in Development & Disease 163

Central to understanding the role of the E2F family of transcription factors in DNA repair has been the identification of a large number of putative and demonstrated E2F target genes. Although E2F proteins were originally characterized as important regulators of cell cycle progression, genome-wide screens have demonstrated much broader roles in a variety of primary and immortalized cell types. For example, E2F-1 and E2F-3 bind to the promoters of apurinic/apyrimidinic endonuclease (APE) and other repair enzymes in human primary epidermal keratinocytes, irrespective of their differentiation status (Chang et al., 2006). Similarly, in the GM06990 lymphoblastoid cell line, non-biased genome-wide screening has identified a large number of putative E2F-4 targets involved in responses to DNA damage (Lee et al., 2011). E2F targets important for DNA repair have also been identified in neoplastic cells following therapeutic intervention. For example, treatment of prostate cancer cells with histone deacetylase inhibitors reduces their ability to repair DNA damage induced by radio- and chemotherapy, thus reducing tumour mass (Kachhap et al., 2010). The impaired ability to repair DNA of treated cells was due, at least in part, to decreased recruitment to and activation by E2F-1 to the promoters of key DNA repair genes. Hence, the importance of E2F factors in DNA repair encompasses not only events during

In spite of the vast similarities in the activities of distinct E2F proteins and their ability to bind potential target Genes, to-date E2F1 is the principal E2F member shown to participate in cellular responses to DNA damage (Bracken et al., 2004). The role of E2F-1 upon DNA damage depends on cellular context. E2F-1 can either induce pro-apoptotic or anti-apoptotic outcomes. During the latter, E2F-1 can play roles to induce cell cycle arrest and upregulate DNA repair, by directing expression of multiple genes. These genes are involved in mismatch repair (MSH2, MLH1), nucleotide excision repair (DDB2, RPA), homologous recombination repair (RAD51, RAD54, RECQL), base excision repair (UNG, APE) & non-homologous end joining (Chang et al., 2006; Ishida et al., 2001; Polager and Ginsberg, 2008; Prost et al., 2007).

In humans, E2F-1 is a 437 amino acid protein, which shows constitutive and rapid nucleocytoplasmic shuttling in a variety of cells (Ivanova et al., 2007). E2F-1 stimulates cell proliferation by positively modulating transcription of genes necessary for DNA synthesis and cell cycle progression (Ivanova et al., 2005). In an apparently paradoxical manner, E2F-1 can also induce cell cycle arrest when associated with pRb, or apoptosis, by activating expression of pro-apoptotic genes (Polager and Ginsberg, 2008). The breadth of E2F-1 targets mediates the distinct biological activities of this transcription factor, which encompass both oncogenic and anti-oncogenic properties, as well as positive modulation of tissue regeneration after injury (D'Souza et al., 2002; Field et al., 1996).

#### **2. E2F-1 and the DNA damage response**

Genotoxic stress in cells activates the DNA damage response, and can occur as a result of a variety of insults. The latter include DNA double- strand breaks and single-strand damage. DNA damage can result from exogenous agents (e.g. radiation, exposure to reactive and mutagenic chemicals), or from endogenous products of cell metabolism (Shiloh, 2003). In response to DNA damage, cells activate multiple pathways that result in apoptosis or in DNA repair, cell cycle arrest, changes in gene expression, as well as in protein synthesis and degradation.

Cells require efficient response mechanisms to genotoxic stress, as this is a life-threatening event because it can significantly alter their genetic material. Multiple mechanisms have evolved to repair damage induced by genotoxic stress, including activation of a global signalling network termed the DNA damage response (DDR), which is capable of detecting distinct types of DNA damage, coordinating appropriate responses. The latter include transcriptional activation, cell cycle arrest, apoptosis, senescence and DNA repair (Shiloh, 2003). The DNA damage response plays a critical role in cell survival when damage occurs during DNA replication. In addition, there are specialized processes, including baseexcision repair (BER), nucleotide-excision repair (NER) & nonhomologous end-joining, which recognize and repair specific types of lesions (Shiloh, 2003). Central to transduce signals that indicate DNA damage and initiate appropriate cellular responses are two related protein kinases, termed ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM can associate with its regulator, the MRN (Mre11-Rad50-NMS1) complex, when double-strand breaks (DSB) are generated (Levitt and Hickson, 2002). On the other hand, ATR forms complexes with its regulator ATRIP (ATR-interacting protein), which senses single-strand DNA (ssDNA) breaks generated by processing of double-strand breaks, as well as single-strand DNA which arises from stalled replication forks (Shiloh, 2003). These two kinases also phosphorylate E2F-1, thus initiating transcriptional activation of its target DNA repair genes.

In spite of the vast similarities in the activities of distinct E2F proteins and their ability to bind potential target Genes, to-date E2F1 is the principal E2F member shown to participate in cellular responses to DNA damage (Bracken et al., 2004). The role of E2F-1 upon DNA damage depends on cellular context. E2F-1 can either induce pro-apoptotic or anti-apoptotic outcomes. During the latter, E2F-1 can play roles to induce cell cycle arrest and upregulate DNA repair, by directing expression of multiple genes. These genes are involved in mismatch repair (MSH2, MLH1), nucleotide excision repair (DDB2, RPA), homologous recombination repair (RAD51, RAD54, RECQL), base excision repair (UNG, APE) & non-homologous end joining

In humans, E2F-1 is a 437 amino acid protein, which shows constitutive and rapid nucleocytoplasmic shuttling in a variety of cells (Ivanova et al., 2007). E2F-1 stimulates cell proliferation by positively modulating transcription of genes necessary for DNA synthesis and cell cycle progression (Ivanova et al., 2005). In an apparently paradoxical manner, E2F-1 can also induce cell cycle arrest when associated with pRb, or apoptosis, by activating expression of pro-apoptotic genes (Polager and Ginsberg, 2008). The breadth of E2F-1 targets mediates the distinct biological activities of this transcription factor, which encompass both oncogenic and anti-oncogenic properties, as well as positive modulation of

Genotoxic stress in cells activates the DNA damage response, and can occur as a result of a variety of insults. The latter include DNA double- strand breaks and single-strand damage. DNA damage can result from exogenous agents (e.g. radiation, exposure to reactive and mutagenic chemicals), or from endogenous products of cell metabolism (Shiloh, 2003). In response to DNA damage, cells activate multiple pathways that result in apoptosis or in DNA repair, cell cycle arrest, changes in gene expression, as well as in protein synthesis and

Cells require efficient response mechanisms to genotoxic stress, as this is a life-threatening event because it can significantly alter their genetic material. Multiple mechanisms have evolved to repair damage induced by genotoxic stress, including activation of a global signalling network termed the DNA damage response (DDR), which is capable of detecting distinct types of DNA damage, coordinating appropriate responses. The latter include transcriptional activation, cell cycle arrest, apoptosis, senescence and DNA repair (Shiloh, 2003). The DNA damage response plays a critical role in cell survival when damage occurs during DNA replication. In addition, there are specialized processes, including baseexcision repair (BER), nucleotide-excision repair (NER) & nonhomologous end-joining, which recognize and repair specific types of lesions (Shiloh, 2003). Central to transduce signals that indicate DNA damage and initiate appropriate cellular responses are two related protein kinases, termed ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM can associate with its regulator, the MRN (Mre11-Rad50-NMS1) complex, when double-strand breaks (DSB) are generated (Levitt and Hickson, 2002). On the other hand, ATR forms complexes with its regulator ATRIP (ATR-interacting protein), which senses single-strand DNA (ssDNA) breaks generated by processing of double-strand breaks, as well as single-strand DNA which arises from stalled replication forks (Shiloh, 2003). These two kinases also phosphorylate E2F-1, thus initiating transcriptional activation

(Chang et al., 2006; Ishida et al., 2001; Polager and Ginsberg, 2008; Prost et al., 2007).

tissue regeneration after injury (D'Souza et al., 2002; Field et al., 1996).

**2. E2F-1 and the DNA damage response** 

degradation.

of its target DNA repair genes.
