**1. Introduction**

The human genome is constantly subjected to DNA damage derived from endogenous and exogenous sources. Normal cellular metabolism can give raise to DNA damage through free radicals production and replication errors, whereas environmental agents, such as ultraviolet (UV) and ionizing radiation (IR), induce specific types of lesions. DNA damage can ultimately lead to genomic instability and carcinogenesis if not properly addressed, thus an elaborate network of proteins has evolved in cells to maintain genome integrity through a pathway termed the DNA-damage response (DDR). DDR allows DNA damage detection, signal propagation and transduction to a multitude of effector proteins, which promote cell survival and activate cell cycle arrest to allow DNA repair. When cells are unable to properly repair DNA, apoptosis or senescence pathways may be triggered, thus eliminating the possibility of passing on damaged or unrepaired genetic material to its progeny. The ultimate goal of DDR is to protect the integrity of genetic information and its faithful transmission, either to DNA by replication or to mRNA by transcription. Therefore, dysregulation of DDR pathway can contribute to carcinogenesis and developmental defects. Ionizing radiation represents a mutagen agent to which human population is exposed due to environmental, professional or accidental reasons. The biological effects of IR depend on the quality and the dose of radiation and on the cell type. Linear energy transfer (LET) represents the energy lost per unit distance as an ionizing particle travels through a material, and it is used to quantify the effects of IR on biological specimens. High-LET radiation (i.e. alpha-particles, neutrons, protons) are densely IR since they lose the energy throughout a small distance, causing dense ionization along their track with high localized multiple DNA damage. Low-LET radiation, such as X and -rays, are sparsely IR since they produce ionizations sparsely along their track and, hence, almost homogeneously within a cell. The biological effect of high-LET radiations are in general much higher than those of low-LET radiations with the same energy. This is because high-LET radiation deposits most of its energy within the volume of one cell and the damage to DNA is therefore larger (Anderson et al., 2002; Brenner & Ward, 1992; Prise et al., 2001). Radiation is potentially harmful to humans, because the ionization it produces can significantly alter the structure of molecules within a living cell.The exposure to ionizing radiation elicits a complex cell response to overcome the dangerous effects of DNA-radiation interaction, such as reactive oxygen species (ROS) production, base oxidation and DNA breaks formation (i.e. single-

The DNA-Damage Response to Ionizing Radiation in Human Lymphocytes 5

promoting the maturation of DSB-associated chromatin (Huen et al., 2007; Mailand et al., 2007; Kolas et al., 2007; Wang et al., 2007). Through its direct interaction with MDC1, RNF8 is recruited to DSB sites along with the other factors in the initial wave of protein accumulation at IRIF (Mailand et al., 2007). Here, RNF8 initiates a complex and tightly regulated ubiquitylation cascade of histones H2A and H2AX at the DSB-flanking chromatin, which causes chromatin restructuring (through incompletely understood mechanisms) associated with the generation of binding sites for protein complexes that accumulate downstream of these early factors (Huen et al., 2007; Mailand et al., 2007).The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. Sumoylation is a signalling mechanism which, analogous to and in parallel with ubiquitination, plays an important role in chromatin remodelling at DSB sites. Sumoylation is catalyzed by SUMOspecific E1, E2, E3s and is reversed by a family of Sentrin/SUMO-specific proteases, SENPs. The SUMO E3 ligases PIAS1 and PIAS4 are required for recruitment of proteins BRCA1 and 53BP1 to IRIF, respectively, and both SUMO1 and SUMO2/3 accumulate at IRIF (Galanty et al., 2009; Morris et al., 2009). Moreover, replicating protein A (RPA70) sumoylation facilitates recruitment of RAD51 to the DNA damage foci to initiate DNA repair through

homologous recombination (Dou et al., 2010).

**mutations in irradiated human lymphocytes** 

rays and low-energy protons.

**2. Cellular effects of ionizing radiation in human lymphocytes** 

**2.1 Surviving fraction,** *HPRT* **mutant frequency and molecular characterization of** 

To contribute to the understanding of the DDR pathway following radiation-induced damage, we studied the effects of IR on human peripheral blood lymphocytes (PBL) irradiated *in vitro* with different doses of -rays and low-energy protons (0.88 MeV; LET: 28keV/m). Irradiated PBL were assayed for cell viability, for mutant frequency at the hypoxanthine-guanine phosphoribosyl transferase (*HPRT*) gene, and for molecular characterization of mutations. The *HPRT* gene, which in humans covers 44 kb and encodes a non-essential protein, allows a wide variety of mutations, from point mutation to total gene deletion, to be detected by using the *HPRT* mutation assay. Deletion of DNA segments is the predominant form of radiation damage in cells that survive irradiation and the mechanisms for producing deletion mutations appear to be very complex and dependent on target cell, gene studied, dose, dose-rate and radiation quality (Schwartz et al., 2000). Large deletions are thought to derive from two DNA double strand breaks close enough to interact each other. Thus, deletion frequency should be dependent on radiation dose and dose-rate. All PBL samples, irradiated either with -rays or protons, showed a dose-dependent cell survival decrease and a *HPRT* mutant frequency increase. In Table 1 we report the data of survival and *HPRT* mutant frequency in human PBL irradiated with different doses of -

Molecular analyses of *HPRT* mutants were carried out in clones derived from PBL exposed to -rays (1-4 Gy) and to low-energy protons (0.5-2Gy), and in non-irradiated clones of the same donors. Among the mutant clones obtained from -irradiated PBL, point mutations were the only kind of mutation in 1Gy irradiated clones, whereas deletions were the prevalent mutations among clones irradiated at 4Gy. In contrast, no partial or total deletions of the *HPRT* gene were detected in mutant clones isolated after proton irradiation. Figure 1

strand breaks, SSBs and double-strand breaks, DSBs ). In particular, DSBs represent the most severe form of damage, since an inefficient or inaccurate repair may lead to cell death or genomic instability (Wyman & Kanaar, 2006). The presence of DSBs leads to a cascade of post-translational modifications of a wide variety of proteins, including phosphorylation, ubiquitinylation, sumoylation, poly(ADP-ribosylation), acetylation and methylation (Huen & Chen, 2010). The early DSB response utilizes phosphorylation-dependent protein–protein interactions to coordinate DNA damage recognition and signal amplification. Following DSB formation the histone H2AX, a histone H2A variant that comprises 10-15% of total cellular H2A in higher eukaryotes, is rapidly phosphorylated on its serine residues 139 (H2AX) (Rogakou et al., 1998) by members of the phosphatidylinositol-3-OH kinase (PI(3)K)-like family, such as ataxia telangiectasia mutated (ATM), DNA-PK and ataxia telangiectasia and Rad3 related (ATR) (Kinner et al., 2008). -H2AX formation occurs within minutes after damage, and extends for up to 1-2 megabases from the site of the break in mammalian cells, providing a platform for subsequent DNA repair protein recruitment and amplification at DSBs (Harper & Elledge, 2007). The phosphorylation of H2AX creates a signal recognized by many proteins of the DNA damage response, which are recruited to the sites of DSBs, forming the ionizing radiation-induced foci (IRIF, Lukas et al., 2004). The biological function of IRIF is thought to shelter the broken DNA ends from decay and prevent illegitimate repair processes, to amplify the DNA damage signal and to provide a local concentration of DDR factors relevant for DNA repair and metabolism. Stabilization of DDR factor recruitment to -H2AX nucleosomes is achieved through the recruitment of a wide variety of proteins regulating ubiquitylation, sumoylation, acetylation, methylation. The mediator of DNA damage checkpoint 1 (MDC1) is the major protein to localize to the sites of DNA breaks in a -H2AX-dependent pathway (Riches et al., 2008; Stucki, 2009) MDC1 has a role in controlling the assembly of multiple repair factors at DNA breaks and in amplifying the DNA damage signal. MDC1 orchestrates the recruitment of IRIF-associated proteins, specifically the MRN complex (MRE11, RAD51, NBS1) and many DNA damage repair proteins, including p53-binding protein 1 (53BP1) and BRCA1 (breast cancer 1). DDR is characterized by the synthesis of ubiquitin conjugates at the sites of damage-induced repair foci (Tanq & Greenberg, 2010). Recently, there has been intense interest regarding the role of ubiquitin and ubiquitin-like molecules in DNA damage repair and signalling, along with its interplay with phosphorylation (Al-Hakim et al., 2010). Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response, in particular in concentrating DNA repair proteins at the sites of DNA damage. The ubiquitylation cascade involves the activities of at least three enzymes: (i) the ubiquitin-activating enzyme (E1); (ii) the ubiquitin-conjugating enzyme (E2); and (iii) the ubiquitin ligase (E3) (Ciechanover et al.,1982; Hershko et al., 1983). E1 employs ATP to adenylate ubiquitin at its C-terminus, which then forms a thioester bond with the E1 activesite cysteine. The modified ubiquitin is then passed on to the E2 enzyme to form another thioester intermediate (the E2∼Ub). Finally, ubiquitin is conjugated to its substrate with the aid of an E3 ubiquitin ligase (Al Hakim et al., 2010). The first E3 ubiquitin ligase that acts in this cascade is RING finger protein 8 (RNF8), which accumulates at DSBs via phosphodependent interactions between its N-terminal fork head associated (FHA) domain and ATM-phosphorylated TQXF motifs on MDC1 (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007). At damaged chromatin, RNF8 cooperates with the E2 conjugating enzyme UBC13 to ubiquitylate histones that likely include H2A and H2AX (Huen, et al., 2007; Mailand et al., 2007, Wu et al., 2008). The ubiquitin ligase RNF8 plays an instrumental role in

strand breaks, SSBs and double-strand breaks, DSBs ). In particular, DSBs represent the most severe form of damage, since an inefficient or inaccurate repair may lead to cell death or genomic instability (Wyman & Kanaar, 2006). The presence of DSBs leads to a cascade of post-translational modifications of a wide variety of proteins, including phosphorylation, ubiquitinylation, sumoylation, poly(ADP-ribosylation), acetylation and methylation (Huen & Chen, 2010). The early DSB response utilizes phosphorylation-dependent protein–protein interactions to coordinate DNA damage recognition and signal amplification. Following DSB formation the histone H2AX, a histone H2A variant that comprises 10-15% of total cellular H2A in higher eukaryotes, is rapidly phosphorylated on its serine residues 139 (H2AX) (Rogakou et al., 1998) by members of the phosphatidylinositol-3-OH kinase (PI(3)K)-like family, such as ataxia telangiectasia mutated (ATM), DNA-PK and ataxia telangiectasia and Rad3 related (ATR) (Kinner et al., 2008). -H2AX formation occurs within minutes after damage, and extends for up to 1-2 megabases from the site of the break in mammalian cells, providing a platform for subsequent DNA repair protein recruitment and amplification at DSBs (Harper & Elledge, 2007). The phosphorylation of H2AX creates a signal recognized by many proteins of the DNA damage response, which are recruited to the sites of DSBs, forming the ionizing radiation-induced foci (IRIF, Lukas et al., 2004). The biological function of IRIF is thought to shelter the broken DNA ends from decay and prevent illegitimate repair processes, to amplify the DNA damage signal and to provide a local concentration of DDR factors relevant for DNA repair and metabolism. Stabilization of DDR factor recruitment to -H2AX nucleosomes is achieved through the recruitment of a wide variety of proteins regulating ubiquitylation, sumoylation, acetylation, methylation. The mediator of DNA damage checkpoint 1 (MDC1) is the major protein to localize to the sites of DNA breaks in a -H2AX-dependent pathway (Riches et al., 2008; Stucki, 2009) MDC1 has a role in controlling the assembly of multiple repair factors at DNA breaks and in amplifying the DNA damage signal. MDC1 orchestrates the recruitment of IRIF-associated proteins, specifically the MRN complex (MRE11, RAD51, NBS1) and many DNA damage repair proteins, including p53-binding protein 1 (53BP1) and BRCA1 (breast cancer 1). DDR is characterized by the synthesis of ubiquitin conjugates at the sites of damage-induced repair foci (Tanq & Greenberg, 2010). Recently, there has been intense interest regarding the role of ubiquitin and ubiquitin-like molecules in DNA damage repair and signalling, along with its interplay with phosphorylation (Al-Hakim et al., 2010). Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response, in particular in concentrating DNA repair proteins at the sites of DNA damage. The ubiquitylation cascade involves the activities of at least three enzymes: (i) the ubiquitin-activating enzyme (E1); (ii) the ubiquitin-conjugating enzyme (E2); and (iii) the ubiquitin ligase (E3) (Ciechanover et al.,1982; Hershko et al., 1983). E1 employs ATP to adenylate ubiquitin at its C-terminus, which then forms a thioester bond with the E1 activesite cysteine. The modified ubiquitin is then passed on to the E2 enzyme to form another thioester intermediate (the E2∼Ub). Finally, ubiquitin is conjugated to its substrate with the aid of an E3 ubiquitin ligase (Al Hakim et al., 2010). The first E3 ubiquitin ligase that acts in this cascade is RING finger protein 8 (RNF8), which accumulates at DSBs via phosphodependent interactions between its N-terminal fork head associated (FHA) domain and ATM-phosphorylated TQXF motifs on MDC1 (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007). At damaged chromatin, RNF8 cooperates with the E2 conjugating enzyme UBC13 to ubiquitylate histones that likely include H2A and H2AX (Huen, et al., 2007; Mailand et al., 2007, Wu et al., 2008). The ubiquitin ligase RNF8 plays an instrumental role in promoting the maturation of DSB-associated chromatin (Huen et al., 2007; Mailand et al., 2007; Kolas et al., 2007; Wang et al., 2007). Through its direct interaction with MDC1, RNF8 is recruited to DSB sites along with the other factors in the initial wave of protein accumulation at IRIF (Mailand et al., 2007). Here, RNF8 initiates a complex and tightly regulated ubiquitylation cascade of histones H2A and H2AX at the DSB-flanking chromatin, which causes chromatin restructuring (through incompletely understood mechanisms) associated with the generation of binding sites for protein complexes that accumulate downstream of these early factors (Huen et al., 2007; Mailand et al., 2007).The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. Sumoylation is a signalling mechanism which, analogous to and in parallel with ubiquitination, plays an important role in chromatin remodelling at DSB sites. Sumoylation is catalyzed by SUMOspecific E1, E2, E3s and is reversed by a family of Sentrin/SUMO-specific proteases, SENPs. The SUMO E3 ligases PIAS1 and PIAS4 are required for recruitment of proteins BRCA1 and 53BP1 to IRIF, respectively, and both SUMO1 and SUMO2/3 accumulate at IRIF (Galanty et al., 2009; Morris et al., 2009). Moreover, replicating protein A (RPA70) sumoylation facilitates recruitment of RAD51 to the DNA damage foci to initiate DNA repair through homologous recombination (Dou et al., 2010).
