**2. DNA double-strand break repair through non-homologous end-joining pathway**

#### **2.1 Homologous Recombination and Non-Homologous End-Joining**

HR is a reaction wherein the genetic material is exchanged between two similar or identical strands of DNA. In the repair of DSB through HR, undamaged DNA serves as a template to reconsitute the original sequence across the break. On the other hand, NHEJ is the direct rejoining of the broken DNA ends without much regard for homology at these ends.

DNA Double-Strand Break Repair Through

Fig. 1. Repair of DSB through NHEJ.

Non-Homologous End-Joining: Recruitment and Assembly of the Players 479

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).

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, nevertheless, important especially in vertebrates.

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 NHEJ can operate.

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 the vertebrate genome.

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 immunodeficiency.

#### **2.2 Processes of NHEJ**

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).
