**2.3.3 XRCC4-DNA ligase IV**

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,

DNA Double-Strand Break Repair Through

(Tsai et al., 2007).

ligase activity.

**2.3.5 Processing enzymes** 

Non-Homologous End-Joining: Recruitment and Assembly of the Players 483

length of DNA (Lu et al., 2007a) and ability to ligate mismatched and non-cohesive ends

Pathologic and physiologic breaks create incompatible DNA ends which are not as easy to rejoin as those created *in vitro* by restriction enzyme digestion. It requires removal of excess DNA and fill-in of gaps and overhangs in order to make them compatible for the DNA

Artemis was identified as the causative gene for human RS-SCID (Moshous et al., 2001). Artemis forms a complex with DNA-PKcs and expresses 5' to 3' exonuclease activity and endonuclease and endonuclease activity at the junction of single-stranded and doublestranded DNA (Ma et al., 2002). Although, the signal joint formation during V(D)J recombination does not require Artemis or DNA-PKcs for joining, all of the components of NHEJ including Artemis are required for coding ends. Artemis in association with DNA-PKcs is deemed necessary for the opening of hairpin structures (Lu et al., 2007b). Artemis is phosphorylated both by DNA-PKcs and ATM (Poinsignon et al., 2004; Zhang et al., 2004). Polymerases μ and λ belong to pol X family and might fill gaps and 5'overhangs (Ramadan et al., 2003). Polynucleotide kinase/phosphatase (PNKP) adds phosphate group to 5' hydroxyl end and also removes phosphate group from 3'-phosphorylated end (Koch et al., 2004; Clements et al., 2004;Whitehouse et al., 2001). Aprataxin (APTX) is initially identified as the product of the gene defective in genetic disorder early-onset ataxia with oculomoter apraxia (Date et al., 2001) and later shown to remove AMP from abortive intermediates of ligation (Ahel et al., 2009). PNK- and APTX-like FHA protein (PALF, also known as APLF, C2orf13 or Xip1) has AP endonuclease activity (Kanno et al., 2007; Iles et al., 2007). Recent study showed that APLF also has histone chaperone activity (Mehrotra et al., 2011) and that it co-operates with PARP-3, which is newly found as a DSB sensor (Rulten et al., 2011). It might be noted that all of these factors bears BRCT or FHA domain as module to bind phosphorylated proteins. Polymerases μ and λ possess BRCT domain. PNKP, APTX and PALF possess FHA domain, which is structurally similar to each other and known to

Apart from the classical NHEJ model, there are also studies by several groups highlighting NHEJ as a more sophisticated and complex mechanism involving a cross–talk between

Human genetic disorder, Ataxia Telangiectasia (AT) is caused by mutation in the ATM (Ataxia Telangiectasia mutated) gene and is characterized by chromosomal instability, immunodeficiency, radiosensitivity, defective cell cycle checkpoint activation and predisposition to cancer indicating its responsibility in genome surveillance (Jorgensen and Shiloh, 1996). ATM deficiency causes early embryonic lethality in Ku or DNA-PKcs deficient mice, thus providing NHEJ an independent role for the DNA-PK holoenzyme (Sekiguchi et al., 2001). ATM and Artemis, together with NBS1, Mre11 and 53BP1 function in a subpathway that repairs approximately 10% of DSBs, probably requiring end-processing (Riballo et al., 2004). Another study suggested three parallel, but mutually crosstalking,

pathways including proteins other than DNA-PKcs, Ku, XRCC4-DNA Ligase IV.

interact with CKII-phosphorylated XRCC1 or XRCC4 (see below).

**2.4 Alternative NHEJ pathways** 

**2.4.1 ATM dependent pathway** 

there are some polymorphisms associated with colorectal cancer and childfood leukemia (Bau et al., 2010; Wu et al., 2010). Disruption of either XRCC4 or DNA Ligase IV gene in mice leads to embryonic lethality with a primary defect in neurogenesis and severe neuronal apoptosis (Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998). Mutants of *DNL4* and *LIF1* genes, the yeast orthologue of human DNA Ligase IV and XRCC4, respectively, exhibited a phenotype similar to that of HDF1 and 2 mutants, indicating its role in recombination and repair (Wilson et al., 1997; Teo and Jackson, 1997, 2000).

XRCC4-DNA Ligase IV is a critical complex formed *in vivo* (Critchlow et al., 1997; Grawunder et al., 1997) for the ligation of the broken DNA ends via NHEJ pathway. The presence of XRCC4 stabilize and activates DNA Ligase IV (Grawunder et al., 1997; Bryans et al., 1999) by stimulating its adenylation which is the first chemical step in ligation (Modesti et al., 1999). XRCC4 forms a homodimer and associates with a polypeptide at the C-terminus of DNA Ligase IV (Critchlow et al., 1997; Junop et al., 2000; Sibanda et al., 2001). This interaction is mapped to the central coiled coil domain of XRCC4 and the inter BRCT linker region at the C-terminus of DNA Ligase IV. This region within DNA Ligase IV, termed as the XRCC4-interacting region (XIR) was deemed necessary and sufficient for XRCC4-Ligase IV interaction (Grawunder et al., 1998). Recently a high resolution crystal structure of human XRCC4 bound to the C-terminal tandem BRCT repeat of DNA Ligase IV was reported. It revealed an extensive binding interface formed by helix-loop-helix structure within the inter-BRCT linker region of Ligase IV, as well as significant interactions involving the second BRCT domain that induces a kink in the tail region of XRCC4 (Wu et al., 2009). This interaction was demonstrated as essential to stabilize the interaction between the XIR of DNA Ligase IV and XRCC4, while the first BRCT domain was considerably dispensable.

#### **2.3.4 XLF/ cernunnos**

Although above five factors had been identified by 1998, there were indications of the existence of additional factor essential for mammalian NHEJ. First, 2BN cell line, which is derived from radiosensitive and immunodeficient patient, showed defective NHEJ but all the known NHEJ components were normal. Second, in 2001, NEJ1/LIF2 was identified as a new essential factor of NHEJ in budding yeast (Kegel et al., 2001; Valencia et al., 2001; Ooi et al., 2001; Frank-Vaillant & Marcand, 2001).

XLF was identified in the yeast two hybrid screen for XRCC4 inteacting protein (Ahnesorg et al., 2006) and named XRCC4-like factor, as it was predicted to have 3D structure similar to that of XRCC4. It is also identified as Cernunnos missing in patients with growth retardation, microcephaly, immunodeficiency, increased cellular sensitivity to ionizing radiation and a defective V(D)J recombination (Buck et al, 2006). It is a 33kDa protein with 299 amino acid residues. NHEJ deficient 2BN cells lacked XLF due to a frameshift mutation (Ahnesorg, 2006). XLF was found to be a genuine homologue of Nej1p from budding yeast (Callebaut et al., 2006). XLF was also shown to be conserved across evolution (Hentges et al., 2006) and to be a paralogue of XRCC4 (Callebaut et al., 2006).

Chromatographic analyses established XLF existing as dimer and crystallographic studies demonstrated its interaction through globular head-to-head domain with that of XRCC4 (Andres et al., 2007; Li et al., 2008). Three-dimensional X-ray scattering characterized a tetramer formation of XRCC4, while the XRCC4-XLF interaction was still mediated through glogular head domains which rendered it suitable for DNA alignment and Ligase IV function (Hammel et al., 2010). XLF possesses DNA binding activity dependent on the length of DNA (Lu et al., 2007a) and ability to ligate mismatched and non-cohesive ends (Tsai et al., 2007).
