**5.2 NHEJ (non-homologous end joining) repair pathway**

The classical NHEJ is a pathway that repairs DSBs. This pathway is generally active in all stages of cell cycles. In NHEJ, the breaks ends are ligated without the need of homologous template. This pathway is very prominent in G0 and G1 phases of cell cycles to repair up to 85% DSBs formed by IR. These breaks formed by IR are very complex and contain non ligatable end groups [45–48].

**107**

*Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ…*

The first step of NHEJ is detection of DSBs site by Ku70/80 proteins. Ku70 (69.8 kDa) & Ku80 (82.7 kDa) is an important heterodimeric complex involved in NHEJ pathway [49]. This dimer is a central DNA binding core and helps in binding of broken ends of DNA with higher affinity. This binding leads to the formation of a bridge between two proximal DNA ends which may help in tethering of the broken ends of damaged DNA [50]. This heterodimer has toroid shape with large central ring to accommodate duplex DNA ends [51, 52]. The inner portion of the central ring is lined with positively charged amino acids. These positively charged amino acids interact with phosphodiester backbone of DNA ends in order to safeguard it from nucleolytic degradation. Ku70 and Ku80 contain unique amino (N) and carboxy (C) terminal regions. The N terminus is phosphorylated by DNA-PKcs and last 12 amino acids of carboxy terminal region of Ku80 is required for interaction of DNA-PKcs with Ku heterodimer [53]. The Ku70 proteins is mandatory for chromosomal organization. The carboxy terminal region of Ku70 is involved in chromosomal organization. The carboxy terminus of Ku70 proteins contains SAP domain (SAF-A/B, Acinus, and PIAS) [54, 55]. The binding of Ku protein with DNA leads to the conformational change in the C terminal region of Ku70 and Ku80. This conformational change facilitates interaction of Ku proteins to other proteins such as XLF, DNA-PKcs, Ligase IV complex, XRCC4 and DNA polymerase μ etc. [55–60]. Thus Ku proteins considered as the corner stone of this pathway. The first protein to interact with Ku is DNA-PKcs. It is also involved in tethering of DNA ends at DSBs which further facilitate the recruitment of other repair proteins [61]. The molecular weight of DNA-PKcs is 469 kDa and contains 4128 amino acids and it is largest protein kinase which is specifically activated by binding to duplex DNA [62]. The conserved region in the extreme C -terminus of Ku80 mediates interaction with C-terminus region of DNA-PKcs. The interaction between DNA-PKcs.Ku further allows DNA-PKcs to interact across the DSB by the formation of (DNAPKcs- Ku-DSB complex or DNA-PK) Synaptic complex which serves to tether the broken ends of the DNA [50]. DNA-PKcs has weak serine threonine kinase activity and it is enhanced by DSB ends and Ku proteins. DNA-PKcs has weak serine threonine kinase activity and it is enhanced by DSB ends and Ku proteins. DNA-PKcs are when autophosphorylated leads to the liberation of DNA ends for processing and ligation. There are sixteen site that has been reported as autophosphorylation sites in DNA PKcs [63, 64]. Autophosphorylation of threonine 2609 and serine 2056 cluster play major roles in NHEJ process. It has been reported that radiosensitivity increases when phosphorylation of entire serine 2056 is inhibited whereas DNA ends processing is accelerated when there is phosphorylation at threonine 2609 [60, 63, 65–67]. The endonucleolytic activity of

Artemis and ligation function of Ligase IV also supported by DNA PKcs [68].

The next step after the detection of DNA ends in NHEJ is processing of the DNA termini to remove non ligatable end groups along with other lesions. Breaks in the DNA induces by IR are complex and depending on the nature of breaks require different processing enzymes like Artemis, DNA polymerase μ/λ, PNK etc. [69]. Artemis has 5′-3′ exonuclease activity however upon complex formation with DNA –PK, it acquires endonuclease activity as well. This acquisition of endonuclease activity helps in opening DNA hair pins during V(D)J recombination [70, 71]. In the processing in DNA the gaps induce by IR are filled by DNA polymerase. The enzyme that plays pivotal role in NHEJ are DNA polymerase μ and λ. These are recruited at DSBs sites by complexation with Ku proteins. Both polymerases

*5.2.2 Processing of DNA ends to remove damaged/non-ligatable groups*

*DOI: http://dx.doi.org/10.5772/intechopen.96374*

*5.2.1 Detection of the DSBs and tethering of the DNA ends*

NHEJ pathway carried out in following steps.

*Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ… DOI: http://dx.doi.org/10.5772/intechopen.96374*

#### *5.2.1 Detection of the DSBs and tethering of the DNA ends*

*DNA - Damages and Repair Mechanisms*

chromatid [38–40].

that HR is an error-free pathway. This pathway is known as error-free because it occurs only S and G2 phases of cell cycles. In these phases of cell cycles sister chromatids are more easily available and can be used as template to synthesize new strands of DNA [30]. HR pathway is essential for cell division in higher eukaryotes to prevent recombination between non identical sequences. HR plays an important role in DNA replication for duplicating the genome and also in telomere mainte-

1.At the end of DSBs processing nucleolytic resection occurs to generate 3′ single-strand overhangs with 3-OH ends. This entire process makes use of MRN complex which has 3′ to 5′ exonuclease activity. 3′ single-strand overhangs are

2.Formation of a recombinase filament on the ssDNA ends: The broken DNA ends has 3′ single-stranded region which is coated with single strand binding protein, RPA. This binding of RPA removes secondary structures. After this, BRCA2 replaced RPA with the help of Rad51. Rad51 protein can interact with many ssDNA binding proteins like BRCA2, RPA, PALB2 and RAD52. Rad51 is 339 amino acid proteins that play an important role in homologous recombination of DNA during DSBs. Rad51 protein forms a helical nucleoprotein filament around DNA. The basis for Rad51 nucleoprotein filament formation to explore the homologous sequences on the sister

3.A displacement loop (D-loop) intermediate is formed by strand invasion into homologous sequence. This invasion is prompted by Rad51, which enhances the activity of another protein Rad54B that facilitates D-loop formation by Rad51 in turn. However in meiosis the recipient DNA is similar but not identical homologous chromosome. D-loop is formed between homologous chromo-

4.Formation of holliday junction: The holliday junction is a biological process that can increase genetic diversity by homologous recombination, shifting gene between homologous and nonhomologous chromosome as well as site specific recombination. This process also involved in DNA DSBs repair pathways. D-loop structure is further changed into cross-shaped structure, known as holliday junction. This occurs after adding of new nitrogenous base to 3′ end of invading strand by DNA polymerase enzyme. This process ultimate leads to restoration of DNA strands on homologous chromosome. The junction is resolved after the restoration of lost sequences information and give error free repaired DNA. The double holiday junction model explained the resolution steps can be carried out by formation of two holliday junction to provide cross-

The classical NHEJ is a pathway that repairs DSBs. This pathway is generally active in all stages of cell cycles. In NHEJ, the breaks ends are ligated without the need of homologous template. This pathway is very prominent in G0 and G1 phases of cell cycles to repair up to 85% DSBs formed by IR. These breaks formed by IR are

nance for the recovery of broken replication fork [31–34].

generated by this exonuclease activity [35–37].

some and invading 3′ overhang strand [38, 41].

over and non-crossover products [42–44].

**5.2 NHEJ (non-homologous end joining) repair pathway**

very complex and contain non ligatable end groups [45–48].

NHEJ pathway carried out in following steps.

HR accomplishes through following steps:

**106**

The first step of NHEJ is detection of DSBs site by Ku70/80 proteins. Ku70 (69.8 kDa) & Ku80 (82.7 kDa) is an important heterodimeric complex involved in NHEJ pathway [49]. This dimer is a central DNA binding core and helps in binding of broken ends of DNA with higher affinity. This binding leads to the formation of a bridge between two proximal DNA ends which may help in tethering of the broken ends of damaged DNA [50]. This heterodimer has toroid shape with large central ring to accommodate duplex DNA ends [51, 52]. The inner portion of the central ring is lined with positively charged amino acids. These positively charged amino acids interact with phosphodiester backbone of DNA ends in order to safeguard it from nucleolytic degradation. Ku70 and Ku80 contain unique amino (N) and carboxy (C) terminal regions. The N terminus is phosphorylated by DNA-PKcs and last 12 amino acids of carboxy terminal region of Ku80 is required for interaction of DNA-PKcs with Ku heterodimer [53]. The Ku70 proteins is mandatory for chromosomal organization. The carboxy terminal region of Ku70 is involved in chromosomal organization. The carboxy terminus of Ku70 proteins contains SAP domain (SAF-A/B, Acinus, and PIAS) [54, 55]. The binding of Ku protein with DNA leads to the conformational change in the C terminal region of Ku70 and Ku80. This conformational change facilitates interaction of Ku proteins to other proteins such as XLF, DNA-PKcs, Ligase IV complex, XRCC4 and DNA polymerase μ etc. [55–60]. Thus Ku proteins considered as the corner stone of this pathway. The first protein to interact with Ku is DNA-PKcs. It is also involved in tethering of DNA ends at DSBs which further facilitate the recruitment of other repair proteins [61]. The molecular weight of DNA-PKcs is 469 kDa and contains 4128 amino acids and it is largest protein kinase which is specifically activated by binding to duplex DNA [62]. The conserved region in the extreme C -terminus of Ku80 mediates interaction with C-terminus region of DNA-PKcs. The interaction between DNA-PKcs.Ku further allows DNA-PKcs to interact across the DSB by the formation of (DNAPKcs- Ku-DSB complex or DNA-PK) Synaptic complex which serves to tether the broken ends of the DNA [50].

DNA-PKcs has weak serine threonine kinase activity and it is enhanced by DSB ends and Ku proteins. DNA-PKcs has weak serine threonine kinase activity and it is enhanced by DSB ends and Ku proteins. DNA-PKcs are when autophosphorylated leads to the liberation of DNA ends for processing and ligation. There are sixteen site that has been reported as autophosphorylation sites in DNA PKcs [63, 64]. Autophosphorylation of threonine 2609 and serine 2056 cluster play major roles in NHEJ process. It has been reported that radiosensitivity increases when phosphorylation of entire serine 2056 is inhibited whereas DNA ends processing is accelerated when there is phosphorylation at threonine 2609 [60, 63, 65–67]. The endonucleolytic activity of Artemis and ligation function of Ligase IV also supported by DNA PKcs [68].

#### *5.2.2 Processing of DNA ends to remove damaged/non-ligatable groups*

The next step after the detection of DNA ends in NHEJ is processing of the DNA termini to remove non ligatable end groups along with other lesions. Breaks in the DNA induces by IR are complex and depending on the nature of breaks require different processing enzymes like Artemis, DNA polymerase μ/λ, PNK etc. [69].

Artemis has 5′-3′ exonuclease activity however upon complex formation with DNA –PK, it acquires endonuclease activity as well. This acquisition of endonuclease activity helps in opening DNA hair pins during V(D)J recombination [70, 71]. In the processing in DNA the gaps induce by IR are filled by DNA polymerase. The enzyme that plays pivotal role in NHEJ are DNA polymerase μ and λ. These are recruited at DSBs sites by complexation with Ku proteins. Both polymerases

are recruited to the DSBs site only when they interact with Ku proteins. They both carry out reactions for gap filling and the only difference in them is requirement of template DNA. Polymerase λ is template dependent whereas polymerase μ is not so much dependent on template DNA. After gap filling proteins such as APLF, PNK, WRN etc. remove non-ligatable ends. The APLF removes non-ligatable ends by exonuclease and endonuclease activities. Whereas PNK removes non-ligatable DNA ends by its 3-DNA phosphatase and 5'-DNA kinase activities. WRN is a member of RecQ helicase family and removes non-ligatable DNA ends by DNA dependent ATPase, 3′-5' DNA helicase and 3′-5′ exonuclease activities [72].
