**6. Alternative NHEJ pathways**

Recent studies identified an alternative repair pathway for DNA DSBs and also known as alternative NHEJ (A-NHEJ). The drawback of alternative pathway is that, it is very slow as compare to C-NHEJ [29]. This pathway is only activate when all other repairing pathways fails to repair DSBs. Because of this, A-NHEJ pathway is also considered as backup pathway for NHEJ (B-NHEJ). In A-NHEJ, the broken ends of DNA are ligated by Ligase III and Ligase I [76, 77]. In 2011, Odell ID et al. explored the effectiveness of Ligase III in repairing DSBs. Ligase III is more effective than Ligase I because Ligase III interact with ERCC1 and PARP1. XRCC1 promote efficient base excision repair and PARP involved in base excision and single strands breaks repair [78]. Apart from this, XRCC1 and PARP1 can also be used as biomarkers to sense the repairing process by A-NHEJ pathway [75]. A recent study illustrates the compromising of A-NHEJ pathway by existing by C-NHEJ factors like Ku proteins etc. A-NHEJ is basically a backup pathway which is activated when NHEJ pathway is compromised. NHEJ pathway is compromised due to absence of one or more core component such as DNA Ligase IV, Ku70/Ku80 heterodimer. A-NHEJ requires single stranded DNA at the ends so certain recombination proteins such as MRE 11A and CtIP act in this pathway [79]. Mutation in NHEJ pathway is extremely rare which makes it difficult to understand whether a-NHEJ is standing pathway or the components involved in this pathway also have its utility in replication, recombination or repair. a-NHEJ require pol θ along with poly (ADPribose) polymerase I(PARP), MRN complex and CtIP [79]. A-NHEJ starts when

**109**

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

partner CtIP and for end joining either LIG1 or LIG3 [25].

**6.1 Role of A-NHEJ in leukemia progression**

phosphorylated CtIP stimulates MRN complex for its endonuclease activity which generates 15-100nucleotide 3′ overhangs. NHEJ requires short microhomolgy of 0–4 bp whereas A-NHEJ requires microhomology of <20 bp. The annealing of the two 3'overhangs is stabilized by pol θ that is sealed by DNA ligase I or DNA ligase III. Apart from these functions pol θ also has transferase activity to add nucleotide to provide microhomology that is absent. Insertion of short templates are not necessarily involved with microhomology but also in human lymphoid translocation around (20–50%) [80]. There have been certain evidences that show pol θ activity when long 3'ssDNA tails generated by the process of extensive resection embeds annealed microhomologies. This process generates non-homologous 3'ssDNA tail that is needed to be removed before extension by pol θ [81]. So during A-NHEJ pathway there may be requirement of nuclease activity from other pathways as well as seen in mammalian system in xeroderma pigmentosunm group F (XPF). XPF uses ERCC1 nuclease complex, APLF or Artemis-DNA –PKcs. Therefore it may be noted that proteins required for A-NHEJ are PARP1, the MRN complex and its

There is a possibility that A-NHEJ is slower than NHEJ as seen in class immunoglobulin class switch recombination where missing DNA ligaseIV can be replaced with DNA ligaseI or DNA ligaseIII. This substitution occurs but with tenfold slower kinetics [82, 83]. This substitution of DNA ligaseIV with DNA ligaseI or DNA ligaseIII suggests presence of backup components of such important enzymes but of lower repair efficiency and with slower kinetics. Future work is needed to identify all the differences between NHEJ and A-NHEJ and also the component of A-NHEJ. Not only distinction but also the repair kinetics is also an important point to be taken into consideration. The fine balance between NHEJ and A-NHEJ is also mediated by ataxia telangiectasia mutated - mediated DNA damage response. In the absence of ATM NHEJ is favored. There is extremely rare and lethal for mammals that lack components for NHEJ [84]. Therefore it should be noted that components for A-NHEJ i.e. its enzymes and proteins may have other functions as well apart from being a substitute. So according to Dueva and Iliakis 2013 there are two models through which A-NHEJ is activated. The first one states that A-NHEJ comes into play when NHEJ or HRR which were engaged for the repair of double strand break but failed to complete the process. According to the second model states that A-NHEJ comes into action when either of the process NHEJ or HRR attempted for the repair mechanism but somehow failed [29]. Basically A-NHEJ comes into play as a backup process for NHEJ or HRR with slight differences. When A-NHEJ back up the failure of NHEJ it can occur throughout the cell cycle as NHEJ is active throughout the cell cycle. But when it backs up the shortcomings of HRR it can only occur in S- and G2- phase of the cell cycle. This type of repair pathway contributes to 10–20% of radiation induced DSBs [34, 85]. A-NHEJ basically operates on resected end that inactivates NHEJ and paves way for HRR which truly justifies the dependences of A-NHEJ on certain proteins such as MRN complex, CtIP, BRCA1 [86, 87].

Leukemia and lymphoma are the type of cancer that shows translocation of chromosomes with involvement of A-NHEJ [88, 89]. There is an availability of evidences that show A-NHEJ play active role in erroneous repair of programmed DSBs during V(D)J AND Class Switch Recombination(CSR) [29]. Severe combined immunodeficiency syndrome (SCID) is a disease which occurs due to mutation in DNA repair proteins [90]. SCID like phenotype is observed in murine models that lack RAG proteins [91–93]. These murine models are also seen to develop tumors because of the translocation in Ig locus due to A-NHEJ. A model was also proposed

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

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

phosphorylated CtIP stimulates MRN complex for its endonuclease activity which generates 15-100nucleotide 3′ overhangs. NHEJ requires short microhomolgy of 0–4 bp whereas A-NHEJ requires microhomology of <20 bp. The annealing of the two 3'overhangs is stabilized by pol θ that is sealed by DNA ligase I or DNA ligase III. Apart from these functions pol θ also has transferase activity to add nucleotide to provide microhomology that is absent. Insertion of short templates are not necessarily involved with microhomology but also in human lymphoid translocation around (20–50%) [80]. There have been certain evidences that show pol θ activity when long 3'ssDNA tails generated by the process of extensive resection embeds annealed microhomologies. This process generates non-homologous 3'ssDNA tail that is needed to be removed before extension by pol θ [81]. So during A-NHEJ pathway there may be requirement of nuclease activity from other pathways as well as seen in mammalian system in xeroderma pigmentosunm group F (XPF). XPF uses ERCC1 nuclease complex, APLF or Artemis-DNA –PKcs. Therefore it may be noted that proteins required for A-NHEJ are PARP1, the MRN complex and its partner CtIP and for end joining either LIG1 or LIG3 [25].

There is a possibility that A-NHEJ is slower than NHEJ as seen in class immunoglobulin class switch recombination where missing DNA ligaseIV can be replaced with DNA ligaseI or DNA ligaseIII. This substitution occurs but with tenfold slower kinetics [82, 83]. This substitution of DNA ligaseIV with DNA ligaseI or DNA ligaseIII suggests presence of backup components of such important enzymes but of lower repair efficiency and with slower kinetics. Future work is needed to identify all the differences between NHEJ and A-NHEJ and also the component of A-NHEJ. Not only distinction but also the repair kinetics is also an important point to be taken into consideration. The fine balance between NHEJ and A-NHEJ is also mediated by ataxia telangiectasia mutated - mediated DNA damage response. In the absence of ATM NHEJ is favored. There is extremely rare and lethal for mammals that lack components for NHEJ [84]. Therefore it should be noted that components for A-NHEJ i.e. its enzymes and proteins may have other functions as well apart from being a substitute. So according to Dueva and Iliakis 2013 there are two models through which A-NHEJ is activated. The first one states that A-NHEJ comes into play when NHEJ or HRR which were engaged for the repair of double strand break but failed to complete the process. According to the second model states that A-NHEJ comes into action when either of the process NHEJ or HRR attempted for the repair mechanism but somehow failed [29]. Basically A-NHEJ comes into play as a backup process for NHEJ or HRR with slight differences. When A-NHEJ back up the failure of NHEJ it can occur throughout the cell cycle as NHEJ is active throughout the cell cycle. But when it backs up the shortcomings of HRR it can only occur in S- and G2- phase of the cell cycle. This type of repair pathway contributes to 10–20% of radiation induced DSBs [34, 85]. A-NHEJ basically operates on resected end that inactivates NHEJ and paves way for HRR which truly justifies the dependences of A-NHEJ on certain proteins such as MRN complex, CtIP, BRCA1 [86, 87].

#### **6.1 Role of A-NHEJ in leukemia progression**

Leukemia and lymphoma are the type of cancer that shows translocation of chromosomes with involvement of A-NHEJ [88, 89]. There is an availability of evidences that show A-NHEJ play active role in erroneous repair of programmed DSBs during V(D)J AND Class Switch Recombination(CSR) [29]. Severe combined immunodeficiency syndrome (SCID) is a disease which occurs due to mutation in DNA repair proteins [90]. SCID like phenotype is observed in murine models that lack RAG proteins [91–93]. These murine models are also seen to develop tumors because of the translocation in Ig locus due to A-NHEJ. A model was also proposed

*DNA - Damages and Repair Mechanisms*

*5.2.3 Rejoining of the broken ends of DNA*

**6. Alternative NHEJ pathways**

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

For the completion of DNA repair process, the broken ends of the processed DNA must be rejoined. In NHEJ pathway, the rejoining and ligation step is carried out by Ligase IV, an ATP dependent enzyme. Ligase IV forms phosphodiester bonds between broken ends of DNA and catalyzing the ligation step. After hydrolysis of ATP, covalent linkage of AMP moiety occurs at specific lysine residue in the active site of DNA ligase. After linkage there is release of pyrophosphate [73]. This process releases AMP. Ligase IV has two C-terminal BRCT domains and is separated by a linker region. The linker region of Ligase IV interacts with the alpha helical region of XRCC4 to form an extremely stable complex. Till date, there are no published data on enzymatic activity of XRCC4 in DNA repairing process. XRCC4 is an important mediator for the recruitment of various NHEJ factors to the site of DNA damage and accelerate the process of DNA repair. Previous studies on NHEJ pathway in DNA repair process support the role of XRCC4 in stabilization and enhancement of DNA Ligase IV enzymatic activity [73–75]. DNA Ligase IV rejoins one strand of DNA at a time and simultaneously recruits and activates other repair-

ATPase, 3′-5' DNA helicase and 3′-5′ exonuclease activities [72].

ing proteins that responsible for ligation of opposite strand of DNA.

Recent studies identified an alternative repair pathway for DNA DSBs and also known as alternative NHEJ (A-NHEJ). The drawback of alternative pathway is that, it is very slow as compare to C-NHEJ [29]. This pathway is only activate when all other repairing pathways fails to repair DSBs. Because of this, A-NHEJ pathway is also considered as backup pathway for NHEJ (B-NHEJ). In A-NHEJ, the broken ends of DNA are ligated by Ligase III and Ligase I [76, 77]. In 2011, Odell ID et al. explored the effectiveness of Ligase III in repairing DSBs. Ligase III is more effective than Ligase I because Ligase III interact with ERCC1 and PARP1. XRCC1 promote efficient base excision repair and PARP involved in base excision and single strands breaks repair [78]. Apart from this, XRCC1 and PARP1 can also be used as biomarkers to sense the repairing process by A-NHEJ pathway [75]. A recent study illustrates the compromising of A-NHEJ pathway by existing by C-NHEJ factors like Ku proteins etc. A-NHEJ is basically a backup pathway which is activated when NHEJ pathway is compromised. NHEJ pathway is compromised due to absence of one or more core component such as DNA Ligase IV, Ku70/Ku80 heterodimer. A-NHEJ requires single stranded DNA at the ends so certain recombination proteins such as MRE 11A and CtIP act in this pathway [79]. Mutation in NHEJ pathway is extremely rare which makes it difficult to understand whether a-NHEJ is standing pathway or the components involved in this pathway also have its utility in replication, recombination or repair. a-NHEJ require pol θ along with poly (ADPribose) polymerase I(PARP), MRN complex and CtIP [79]. A-NHEJ starts when

**108**

which suggests A-NHEJ mediated genomic instability was suppressed with the help of RAG1/2 proteins and NHEJ factors [94, 95]. RAG complex formed post cleavage shunts the broken ends of DNA to NHEJ thus suppressing recombination events. It is seen that RAG mediated DSB repair during CSR is not compromised in cells lacking NHEJ but is shifted to A-NHEJ [82, 96, 97]. There is effect of absence of DNA-PKcs and it uses Lig1 or Lig3.XRCC1 which acts together with Lig3 is not necessary for A-NHEJ during CSR. Infect the absence of these components increases CSR efficiency [98, 99]. PARP1 and PARP2 is nonessential component during CSR but PARP1 favors A-NHEJ whereas PARP2 suppress translocation during CSR [100]. It is very interesting to note that in chronic myelogenous leukemia (CML) there is increased production of ROS due to increased cell division which is facilitated by BCR-ABL tyrosine kinase. Increased ROS inside the cells leads to DNA damages especially DSB. This leads to the up-regulation of A-NHEJ [101–103]. The cells which are BCR-ABL positive CML shows up regulation of key proteins for A-NHEJ i.e. Lig3α and WRN whereas down regulation of key proteins of NHEJ Artemis and Lig4. Therefore A-NHEJ enables the cells of CML to repair ROS induced DSB and survive. Though this repair pathway of A-NHEJ is error prone the price the cells pay for survival is genomic instability [104].

In acute myeloid leukemia (AML) mutation that occurs are internal tandem duplication (ITD) of FMS-like tyrosine kinase3 (FLT3) receptor. FLT3-ITD is type of cancer which utilizes microhomology mediated A-NHEJ to repair double strand breaks. It causes increased number of deletion. The cells expressing FLT3-ITD has increased protein level of Lig3α but decreased level of Ku protein required for NHEJ. This causes shift towards the A-NHEJ for DSB repair [105].
