**2.3 Components of NHEJ**

#### **2.3.1 Ku**

Ku protein was initially found as the antigen of autoantibody in a patient of polymyositisscleroderma overlap syndrome (Mimori et al., 1981). Biochemical approach, including immunoprecipitation of [32P]orthophosphate or [35S]methionine-labeled cell extract and

Therefore, NHEJ may sometimes incur nucleotide deletions or insertions at the junction or joining with incorrect partner, leading to chromosomal abberations like duplications, inversions or translocations. Hence it is considered that NHEJ is less accurate than HR but,

In HR, the template should be found in homologous chromosome or in sister chromatid. Organisms like budding yeast can avail homologous chromosome as the template. However, vertebrate can utilize only sister chromatid, but not homologous chromosome, as the template for HR and, therefore, the repair of DSB through HR is limited to late S and G2 phases. The majolity of the cells reside in G0 or G1 phases in vertebrate body, where only

Additionally, only small portions of the genome in vertebrate are encoding protein or functional RNA and other portions are intervening or repetitive sequences. These regions may have important roles in the structural maintenance of the genome, proper replication/segregation of the genome or spatiotemporal regulation of the genen expression. Nevertheless, small deletion or insertion of nucleotides might be tolerated in most portion of

Finally, whereas HR is utilized in meiotic recombination in reproductive organs, NHEJ is utilized in V(D)J recombination in immune system to establish diversity of immunogloblins and T cell recepters. Thus, genetic defect in either one of NHEJ components results not only in elevated sensitivity toward radiation and radiomimetic agents but also in

NHEJ process may be divided into three steps, i.e., (i) detection, (ii) processing and (iii) ligation of DSB ends (Fig.1). The detection and ligation steps comprises the core reaction while the processing step is required only when the ends are not readily ligatable. In the detection step, Ku protein, heterodimer consisting of Ku70 and Ku86 (also known as Ku80), first binds to the ends of double-stranded DNA and then recruits DNA-PK catalytic subunit (DNA-PKcs). The complex consisting of Ku70, Ku86 and DNA-PKcs is termed DNAdependent protein kinase (DNA-PK). Upon binding of DNA-PKcs to DNA ends, it exerts kinase catalytic activity to phosphorylate substrate proteins. Thus, DNA-PK is considered the molecular sensor of DSB, triggering the signalling cascade. At the final ligation step, DNA ligase IV in a tight association with XRCC4 catalyzes the reaction to join the two DNA ends. XRCC4-like factor, XLF, which is also known as Cernunnos, is also essential at this step, especially when two ends are not compatible. Thus, six polypeptides, i.e., Ku70, Ku86, DNA-PKcs, DNA ligase IV, XRCC4 and XLF are core components of NHEJ. Processing step might involve a number of enzymes depending on the shape of each DNA end and compatibility of two ends to be ligated. Presumed processing enzymes contain Artemis, DNA polymerase μ/λ, polynucleotide kinase/phosphatase (PNKP), Aprataxin (APTX) and

Aprataxin and PNKP-like factor (APLF, also known as PALF, C2orf13 or Xip1).

Ku protein was initially found as the antigen of autoantibody in a patient of polymyositisscleroderma overlap syndrome (Mimori et al., 1981). Biochemical approach, including immunoprecipitation of [32P]orthophosphate or [35S]methionine-labeled cell extract and

nevertheless, important especially in vertebrates.

NHEJ can operate.

the vertebrate genome.

immunodeficiency.

**2.2 Processes of NHEJ** 

**2.3 Components of NHEJ** 

**2.3.1 Ku** 

immunoaffinity purification, lead to identify Ku as a DNA-binding protein made up of two subunits of 70,000Da and 80,000Da, respectively, which are now known as Ku70 and Ku80 (or Ku86) (Mimori et al., 1986). It is also estimated that Ku is an abundant protein, existing as 400,000 copies in logarithmically growing HeLa cells. Protein-DNA interaction studies, including footprint analysis, led to the finding that Ku binds to the ends of double-stranded DNA without requirement for specific sequence (Mimori & Hardin, 1986). Because of this striking property, possible role of Ku in DNA repair or in transposition was suspected. In early 1990s, Ku was found to be an essential component of DNA-PK (Dvir et al., 1992, 1993; Gottlieb and Jackson, 1993). It was also found that Ku80 is equivalent to XRCC5 (X-ray repair cross complementing) gene product, which is missing in X-ray sensitive rodent cell lines including *xrs*-5, -6, XR-V9B and XR-V15B (Taccioli et al., 1994; Smider et al., 1994).

DNA Double-Strand Break Repair Through

have defect in Artemis activation (below).

dictyostelium (Hudson et al., 2005).

NHEJ to HR.

**2.3.3 XRCC4-DNA ligase IV** 

*in vivo* phosphorylation target(s) essential for DNA repair.

Non-Homologous End-Joining: Recruitment and Assembly of the Players 481

PKcs is also found in horse (Wiler et al., 1995; Shin and Meek, 1997) and in dog (Meek et al., 2001). M059J, a human glioma cell line, defective in DNA-PKcs, also showed radiosensitivity with defective DSB repair (Lees-Miller et al., 1995). Recently, DNA-PKcs missense mutation was identified in human radiosensitive T- B- severe combined immunodeficiency (TB-SCID) (van der Burg et al., 2009). Cells from the patient exhibit normal DNA-PK activity but may

Cloning of gene revealed that DNA-PKcs is a 4,127 amino acid polypeptide, one of the largest molecules in the cell (Hartley et al., 1995). The carboxy-terminal between amino acid residues 3719 – 4127 compose the catalytic domain that is categolized into phosphatidylinositol-3 kinase and like kinase (PIKK) family (Hartley et al., 1995; Poltoratsky et al., 1995). PIKK family include ataxia-telangiectasia mutated (ATM) (Savitsky et al., 1995) and ATM- and Rad3-related (ATR) (Cimprich et al., 1996), both of which are protein kinases with roles in DNA repair and cell cycle checkpoint as sensors of DNA damages. Although orthologues of ATM and ATR can be found in fruit fly, nematoda, plants and yeast, DNA-PKcs has been found only in vertebrate, some arthropods (Dore et al., 2004) and

*In vitro* studies had revealed that DNA-PK can phosphorylate a number of nuclear, DNA binding proteins with supposed functions in transcription, replication, recombination and repair (Lees-Miller et al., 1992). The sites phosphorylated by DNA-PK were identified as serine and threonine that are immediately followed by a glutamine on the linear sequence; SQ/TQ (Lees-Miller et al., 1992), although there are a considerable number of exceptions reported. The protein phosphorylation by DNA-PK should be essential for NHEJ, as catalytically inactive form of DNA-PKcs can restore at most partial NHEJ activity to DNA-PKcs deficient cells (Kurimasa et al., 1999). However, it is presently unclear what is/are the

Recent studies have shed light on the phosphorylation of DNA-PKcs itself. At least 16 sites of autophosphorylation have been identified (Chan et al., 2002; Douglas et al., 2002; Ding et al., 2003). Most of them are clustered within 2023 - 2056 (PQR cluster), 2609 – 2647 (ABCDE cluster) and 2671 – 2677. Some of them may be phosphorylated by ATM or ATR *in cellulo* (Chen et al., 2007; Yajima et al., 2006) It has been demonstrated that, *in vitro*, autophosphorylation of DNA-PK leads to loss of kinase activity and dissociation from Ku (Chan et al., 1996). It should be also noted that substitution of serines and threonines within ABCDE cluster with alanine results in greater radiation sensitivity than DNA-PKcs null cells and also in reduced rates of HR. Thus, autophosphorylation, especially within ABCDE cluster might regulate DNA-PK activity negatively or switch repair pathway from

XRCC4 was isolated and cloned from a human cDNA sequence whose expression in the XR-1 cells, derived from Chinese Hamster ovary and phenotypically similar to *scid* and *xrs,* conferred normal V(D)J recombination ability and also DSB repair activity (Li et al., 1995). Biochemical studies lead to finding that it is associated with DNA ligase IV (Critchlow et al., 1997; Grawunder et al., 1997). Mutations in DNA ligase IV gene have been identified in radiosensitive leukemia patient (Badie et al, 1995; Riballo et al., 1999) and in patients exhibiting developmental delay and immunodeficiency, which is called ligase IV syndrome (O'Driscoll et al., 2001). Although mutation in XRCC4 gene has not been found in humans,

These cell lines also exhibit defect in V(D)J recombination, indicating the role of Ku in this process. Ku80 knockout mice showed immunodeficiency and radiosensitivity, like *scid* mice (below) and also exhibited growth defect; body weight was 40-60% of age-matched control (Nussenzweig et al., 1996; Zhu et al., 1996). Ku70 knockout mice also showed immunodeficiency, radiosensitivity and growth defect. However, immunological defect in Ku70 knockout mice was less severe than in Ku80 knockout mice, as it shows partial production and differentiation of T cells (Gu et al., 1997; Ouyang et al., 1997). Ku is also implied to play critical roles in telomere capping in mammalian cells (Hsu et al., 2000).

Homologues of Ku proteins were identified not only in mammals but also in other eukaryota including budding yeast, where it is referred to as HDF1 and HDF2 (high- affinity DNA binding factor), or Yku70 and Yku80, respectively (Feldmann & Winnacker, 1993; Milne et al., 1996; Feldmann et al., 1996; Boulton & Jackson, 1996). Yeast Ku is shown to play important roles in NHEJ, telomere maintenance and silencing (Boulton & Jackson, 1996, 1998; Porter et al., 1996).

Ku70 and Ku80 show low but significant sequence similarity, indicating common evolutional origin, and share a similar structural configuration (Dynan & Yoo, 1998; Gell & Jackson, 1999). Expectedly, "single" Ku orthologue was identified in bacteria and in bacteriophage (Weller et al., 2002; d'Adda di Fagagna et al., 2003). As revealed by X-ray crystallography, Ku 70 and Ku80 fold to form an asymmetric ring shaped structure forming an aperture large enough to let DNA thread through it; thus playing a crucial role in DSB recognition (Walker et al., 2001). The core of Ku required to form dimer and aperture is conserved among all Ku orthologues. Both of Ku70 and Ku80 bear von Willebrant factor A domain, which may be essential for heterodimer formation. The C-termial of Ku70 bears SAP domain, which may mediate DNA binding, and the C-terminal of Ku80 bears a conserved motif to interact with DNA-PKcs (Gell & Jackson, 1999; Falck et al.., 2005).

Ku translocates along DNA in an ATP-independent manner, allowing several dimers to bind on a single DNA molecule (Zhang and Yaneva, 1992; Bliss and Lane, 1997). Ku was identified also as a ssDNA dependent ATPase stimulating the DNA polymerase α primase activity (Vishwanatha and Baril, 1990; Cao et al., 1994) and as an ATP dependent DNA helicase II (HDH II) (Tuteja et al., 1994). Recent study demonstrated that Ku has 5'-RP/AP lyase activity, nicking 3'-side of abasic site (Roberts et al., 2010). Thus, Ku might exert multiple functions, not only binding to DSBs but also activating damage signal via DNA-PKcs and processing DSB ends removing the obstacle for ligation.

#### **2.3.2 DNA-PKcs**

DNA-PK activity was first found as an activity to phosphorylate Hsp90 in the presence of double-stranded DNA in the extracts of HeLa cell, rabbit reticulocyte, Xenopus egg and sea urchin egg (Walker et al., 1985). DNA-PK was purified from Hela cell nuclei as a 300-350 kDa protein, which is now called DNA-PKcs for DNA-PK catalytic subunit (Carter et al., 1990; Lees-Miller et al., 1990). Later it was found that Ku is an essential component of DNA-PK and that DNA-PK requires binding to DNA ends to be activated (Dvir et al., 1992, 1993; Gottlieb and Jackson, 1993). Following the finding that XRCC5 is equivalent to Ku80, DNA-PKcs is found to correspond to XRCC7, which is deficient in *scid* (severe combined immunodeficiency) mouse (Kirchgessner et al., 1995; Blunt et al., 1995; Peterson et al., 1995), lacking mature B and T cells due to a defect in V(D)J recombination (Bosma et al., 1983; Lieber et al., 1988; Fulop & Phillips, 1990; Biederman et al., 1991). Scid due to defect in DNA-

These cell lines also exhibit defect in V(D)J recombination, indicating the role of Ku in this process. Ku80 knockout mice showed immunodeficiency and radiosensitivity, like *scid* mice (below) and also exhibited growth defect; body weight was 40-60% of age-matched control (Nussenzweig et al., 1996; Zhu et al., 1996). Ku70 knockout mice also showed immunodeficiency, radiosensitivity and growth defect. However, immunological defect in Ku70 knockout mice was less severe than in Ku80 knockout mice, as it shows partial production and differentiation of T cells (Gu et al., 1997; Ouyang et al., 1997). Ku is also implied to play critical roles in telomere capping in mammalian cells (Hsu et al., 2000). Homologues of Ku proteins were identified not only in mammals but also in other eukaryota including budding yeast, where it is referred to as HDF1 and HDF2 (high- affinity DNA binding factor), or Yku70 and Yku80, respectively (Feldmann & Winnacker, 1993; Milne et al., 1996; Feldmann et al., 1996; Boulton & Jackson, 1996). Yeast Ku is shown to play important roles in NHEJ, telomere maintenance and silencing (Boulton & Jackson, 1996,

Ku70 and Ku80 show low but significant sequence similarity, indicating common evolutional origin, and share a similar structural configuration (Dynan & Yoo, 1998; Gell & Jackson, 1999). Expectedly, "single" Ku orthologue was identified in bacteria and in bacteriophage (Weller et al., 2002; d'Adda di Fagagna et al., 2003). As revealed by X-ray crystallography, Ku 70 and Ku80 fold to form an asymmetric ring shaped structure forming an aperture large enough to let DNA thread through it; thus playing a crucial role in DSB recognition (Walker et al., 2001). The core of Ku required to form dimer and aperture is conserved among all Ku orthologues. Both of Ku70 and Ku80 bear von Willebrant factor A domain, which may be essential for heterodimer formation. The C-termial of Ku70 bears SAP domain, which may mediate DNA binding, and the C-terminal of Ku80 bears a

conserved motif to interact with DNA-PKcs (Gell & Jackson, 1999; Falck et al.., 2005).

PKcs and processing DSB ends removing the obstacle for ligation.

Ku translocates along DNA in an ATP-independent manner, allowing several dimers to bind on a single DNA molecule (Zhang and Yaneva, 1992; Bliss and Lane, 1997). Ku was identified also as a ssDNA dependent ATPase stimulating the DNA polymerase α primase activity (Vishwanatha and Baril, 1990; Cao et al., 1994) and as an ATP dependent DNA helicase II (HDH II) (Tuteja et al., 1994). Recent study demonstrated that Ku has 5'-RP/AP lyase activity, nicking 3'-side of abasic site (Roberts et al., 2010). Thus, Ku might exert multiple functions, not only binding to DSBs but also activating damage signal via DNA-

DNA-PK activity was first found as an activity to phosphorylate Hsp90 in the presence of double-stranded DNA in the extracts of HeLa cell, rabbit reticulocyte, Xenopus egg and sea urchin egg (Walker et al., 1985). DNA-PK was purified from Hela cell nuclei as a 300-350 kDa protein, which is now called DNA-PKcs for DNA-PK catalytic subunit (Carter et al., 1990; Lees-Miller et al., 1990). Later it was found that Ku is an essential component of DNA-PK and that DNA-PK requires binding to DNA ends to be activated (Dvir et al., 1992, 1993; Gottlieb and Jackson, 1993). Following the finding that XRCC5 is equivalent to Ku80, DNA-PKcs is found to correspond to XRCC7, which is deficient in *scid* (severe combined immunodeficiency) mouse (Kirchgessner et al., 1995; Blunt et al., 1995; Peterson et al., 1995), lacking mature B and T cells due to a defect in V(D)J recombination (Bosma et al., 1983; Lieber et al., 1988; Fulop & Phillips, 1990; Biederman et al., 1991). Scid due to defect in DNA-

1998; Porter et al., 1996).

**2.3.2 DNA-PKcs** 

PKcs is also found in horse (Wiler et al., 1995; Shin and Meek, 1997) and in dog (Meek et al., 2001). M059J, a human glioma cell line, defective in DNA-PKcs, also showed radiosensitivity with defective DSB repair (Lees-Miller et al., 1995). Recently, DNA-PKcs missense mutation was identified in human radiosensitive T- B- severe combined immunodeficiency (TB-SCID) (van der Burg et al., 2009). Cells from the patient exhibit normal DNA-PK activity but may have defect in Artemis activation (below).

Cloning of gene revealed that DNA-PKcs is a 4,127 amino acid polypeptide, one of the largest molecules in the cell (Hartley et al., 1995). The carboxy-terminal between amino acid residues 3719 – 4127 compose the catalytic domain that is categolized into phosphatidylinositol-3 kinase and like kinase (PIKK) family (Hartley et al., 1995; Poltoratsky et al., 1995). PIKK family include ataxia-telangiectasia mutated (ATM) (Savitsky et al., 1995) and ATM- and Rad3-related (ATR) (Cimprich et al., 1996), both of which are protein kinases with roles in DNA repair and cell cycle checkpoint as sensors of DNA damages. Although orthologues of ATM and ATR can be found in fruit fly, nematoda, plants and yeast, DNA-PKcs has been found only in vertebrate, some arthropods (Dore et al., 2004) and dictyostelium (Hudson et al., 2005).

*In vitro* studies had revealed that DNA-PK can phosphorylate a number of nuclear, DNA binding proteins with supposed functions in transcription, replication, recombination and repair (Lees-Miller et al., 1992). The sites phosphorylated by DNA-PK were identified as serine and threonine that are immediately followed by a glutamine on the linear sequence; SQ/TQ (Lees-Miller et al., 1992), although there are a considerable number of exceptions reported. The protein phosphorylation by DNA-PK should be essential for NHEJ, as catalytically inactive form of DNA-PKcs can restore at most partial NHEJ activity to DNA-PKcs deficient cells (Kurimasa et al., 1999). However, it is presently unclear what is/are the *in vivo* phosphorylation target(s) essential for DNA repair.

Recent studies have shed light on the phosphorylation of DNA-PKcs itself. At least 16 sites of autophosphorylation have been identified (Chan et al., 2002; Douglas et al., 2002; Ding et al., 2003). Most of them are clustered within 2023 - 2056 (PQR cluster), 2609 – 2647 (ABCDE cluster) and 2671 – 2677. Some of them may be phosphorylated by ATM or ATR *in cellulo* (Chen et al., 2007; Yajima et al., 2006) It has been demonstrated that, *in vitro*, autophosphorylation of DNA-PK leads to loss of kinase activity and dissociation from Ku (Chan et al., 1996). It should be also noted that substitution of serines and threonines within ABCDE cluster with alanine results in greater radiation sensitivity than DNA-PKcs null cells and also in reduced rates of HR. Thus, autophosphorylation, especially within ABCDE cluster might regulate DNA-PK activity negatively or switch repair pathway from NHEJ to HR.
