**8. Interstrand crosslinks lesion repair mechanisms**

Lesions in interstrand crosslinks epitomize an arduous challenge in genome maintenance pathways due to the compromise of genomic information present on both strands. Therefore, an application of non-damaged strand as a template for accurate repair in straightforward cut and patch mechanism is not feasible. In this regard, ICL repair employs the concerted and synchronized interaction of dynamics from numerous mechanisms of DNA damage repair, including NER, homologous recombination, mismatch repair, translesion synthesis, ataxia telangiectasia, Rad3 related and Fanconi anemia pathway. **Figure 1** illustrates the schematic mechanism of ICL repair [75].

#### **Figure 1.**

*DNA - Damages and Repair Mechanisms*

succeeded by Pol ζ that extends the unpaired strand.

**6. ICL lesion removal in quiescent G0/G1 phase**

machinery so that the extension of stalled DNA strand occur on one base before the ICL region [64]. On unwinding, Rev1's deoxycytidyl transferase of Rev1 incorporates cytosine on the complementary strand across the ICL region [65]. This is then

The comprehension of ICL repair is a difficult task because it has an implication on both strands of DNA. The cells in G0/G1 phase do not require homologous recombination for ICL repair [66]. Moreover, all eukaryotic organisms ranging from *Saccharomyces cerevisiae* to the human beings, require NER for the incisions of ICL. The single stranded gap is produced at the first step of NER by the oligonucleotide on ICL lesion. This can be bypassed with the help of translesion DNA polymerases REV1 just like the DNA polymerases (η, ι, κ, and ζ,). Both the DNA

polymerases κ, and ζ, as well as REV1 are vital for this stage of NER [67]**.**

The repair of ICL faces several complications during the S phase. The data exhibits the formation of double stranded breaks by interaction with ICL causing agents [59]. The ICL induced Double stranded breaks can be repaired by HR rather than non-homologous end joining (NHEJ) method [68]. This brings to the conclusion that ICL-induced DSBs are linked with DNA replication forks. NER indicates ICLs in *S. cerevisiae* and NER function is important for ICL repair. So, all NER-mutants exhibit hyper sensitivity to the ICL causative agents. Contrarily, the cells deficient in *XPF*- as well as *ERCC1*- show immense hypersensitivity to the ICL agents (mitomycin C & nitrogen mustard) in mammals. The product of *XPF* as well as *ERCC1* make up an endonuclease which is hetero-dimeric in nature identifies and incise the single stranded branched structures [69]. Moreover, MUS81-EME1 along with XPF-ERCC1, the homologous structure specific endonucleases are also keen in repairing the ICL lesions [70]. MUS81-EME1 is notable for its binding with the double-stranded branched structures, flaps at 3′ end, as well as Holliday junctions [71]. Either of the two XPF-ERCC1 and MUS81-EME1 are responsible for ICL-induced double strand formation. Since, a multitude of nucleases are recognized recently being the key players in ICLs incision, the mechanism underlying the process need to be explored. We abridge the current knowledge about the ICL repair mechanism in S phase. HR repairs the ICLs induced DSBs. An experiment conducted in *S. cerevisiae*, gives an outline of hypersensitivity against ICL causative agents in *rad51*, *rad52, rad54*, *rad59*, as well as *mre11* mutants but not in case of *yku70* mutants. The hypersensitivity of *rad52 yku70* double mutants to ICLs is at par with that of rad52 mutants [72]. The HR deficient strains show the increase in accumulated DSBs successively on treating with ICL inducers as there lacks an ability to cure DSB which means that NHEJ is not a pre-requisite to remediate DSBs stimulated by ICLs. The mammals follow the same process in their cells. The HR deficient cells depict hypersensitivity against ICLs like cells having mutated paralogs of *RAD51*, *RAD54*, *RAD54B*, along with *BRCA2*, while it is not observed in cells deficient in NHEJ [73]. It significantly highlights the role of HR in repairing DSBs and re-initiating the halted replication forks of DNA. Fanconi anemia (FA) genes are key players in the remediation of ICL in eukaryotes. The proper role of FA gene products in biochemical reactions are still not identified properly, but are notable for their control of HR

**7. ICL recognition and repair in proliferating S-phase**

**128**

at the replication forks of DNA [74].

*Schematic of ICL repair mechanism. (1) After the formation of ICLsin the cells, UHRF1 is recruited through its SRA domain immediately. (2) Single replication fork reaches at ICL. (3) Then Replication machinery is transversed through ICL by the help of FANCM/MHF complex and allowes the ICL for later repair. (4), (5) On an alternate basis FANCS or BRCA1 allows the unloading of CMG helicase complex, when second replication fork arrives at ICL. (6) Then replicative polymerase reaches at −1 position of ICL, leaving X shaped similar to the transverse mechanism. (7) Then ATR allows the phosphorylation of FANCD2/FANCI complex at multiple sites and meanwhile FA core complex mono-ubiquitinate at FANCD2/FANCI complex at K561 and K523 respectively. (8) The complex is then recruited to ICL at the replication fork. (9), (10) This ubiquitinated complex recruits SLX4/XPF on ICL in order to unhook the ICL. (11) Afterwards, CtlP an MRN complex resect the double strand breaks and BRCA2 facilitates the formation of RAD51 filament on single stranded DNA generated by resection. (12) Then Polζ carry out the polymerization step through the unhooked ICL. (13) Rad51 then facilitates the invasion of strand with subsequent extension of the other strand. (14) Lastly SLX4 and nucleases resolve the Holliday junction (15) and NER repair proteins remove the damaged nucleotides.*

#### **8.1 Role of homologous recombination in ICL repair**

The phenomenon of homologous recombination repair (HRR) employs homologous DNA sequences as template for repair and tolerance of DNA lesions that obstruct DNA replication in S-phase. Homologous recombination usually encompasses four step (i) double strand break recognition tailed by nucleolytic processing to produce 3′ single stranded ends of DNA, (ii) protein-mediated strand invasion of single-stranded DNA with homologous chromosome (iii) synthesis of DNA which regenerates degraded DNA using undamaged homologous chromosome as a template and (iv) resolution of Holliday junction intermediates. Usually the platinum drugs drive fruitful results in the treatment of BRCA1- and BRCA2- associated ovarian cancers [76]. However, the protein products of these two genes give rise to HR-mediated repair of DNA damage. A dynamic combination of BRCA1 and associated RING domain protein 1 (BARD1) exhibits ubiquitin ligase activity that is essential for the proper localization of RAD51, which is a central player in Homologous Recombination repair. Through BRCA2 mediated interaction with RAD51, it is specifically targeted to sites where recombination is initiated [77]. However, RAD51 deficient cells represent hypersensitivity towards ICL-inducing agents.

In this regard, the model organism, *Escherichia coli* has provided deep insights in the mechanisms involved in HRR of bacteria. Usually, RecA of bacteria has proven to be an effective protein in all major aspects of HRR due to its ability of forming nucleoprotein filament with both single and double stranded DNA. In *E. coli,* RecBCD complex- combination of nuclease/helicase, initiates the phenomenon of recombination by creating 3′-terminal single-stranded DNA substrate for the activity of RecA protein. RecBCD complex usually binds to the end of linear double stranded DNA and RecA in combination with single-stranded binding proteins (SSBP) allows an incessant formation of presynaptic filament on DNA. This nucleoprotein complex allows a rapid and efficient search for homology within the double-stranded DNA recipient, with subsequent formation of a joint molecule. After the formation of joint molecule, DNA PolI regenerates the sequence and the resultant Holliday junction is resolved by the action of RuvC protein that acts in concert with RuvAB proteins to coordinate the steps of branch migration and Holliday junction resolution [78].

In *Saccharomyces cerevisiae*, the incision of DNA is carried out by an anonymous nuclease. A yeast homologue of RecA, Rad51 works in conjunction with Rad52 dislocates the single-stranded DNA that is ostensibly covered by RPA. The subsequent nucleofilament works with Rad54 and Rad55/57 in DNA unwinding and strand annealing between donor DNA and incoming Rad51 nucleoprotein. The resolution of subsequent recombination intermediates is frequently carried out by assorted set of mechanisms including mus81-mms4 nuclease and Resolvase A [79].

#### **8.2 Translesion DNA synthesis in DNA interstrand crosslinks**

Translesion DNA Synthesis polymerases are considered essential for ICL repair in both S/G2 and G1 to bypass an ICL unhooked from one of the two cross-linked strands. The phenomenon of Translesion synthesis encompasses multiple polymerases with a dynamic ability to carry out an insertion of nucleotide across the lesion and others carrying out further extension. Based on genetic and biochemical studies, an assortment of polymerases has been implied in repair of ICLs. Usually translesion synthesis is a threefold step: (i) release of replicative polymerase after an interruption of normal bidirectional DNA with lesion, (ii) release of specialized translesion polymerase onto a site and starts the replication at a short distance past the lesion, (iii) the replacement of translesion polymerase with replicative DNA polymerase which continues the normal process of replication [80].

**131**

*Interstrand Crosslink Repair: New Horizons of DNA Damage Repair*

For HR-mediated repair of replication-dependent DSB and excision of ICL from the genome, this is vital to generate an intact template. In this regard, an assortment of polymerases allows the bypass of unhooked ICLs in vitro by using cross-linked DNA substrate model. In *Escherichia coli,* PolIV can easily bypass the unhooked ICLs


The role of TLS polymerases in ICL repair is strongly supported by the study of genetics. In yeast, mutations in genes encoding subunits of Polζ i.e. Rev3, Rev7 or REV1 render cells hypersensitive to cross-linking agents [72]. Polζ is majorly important for the cross-linking resistance of non-replicating cells. However, to date *in vitro* studies have not been able to show bypass of ICL damage by Pol ζ-REV1, thus suggesting the other factors involved in lesion bypass. However, Pol η mutants

In mammals, Pol ζ (comprising of REV3 and REV7 subunits) and REV1 are significant factors in ICL repair. However, the cells deficient in any of the aforementioned genes are highly sensitive to various cross linking agents [83]. REV1 act as TLS polymerase scaffold and thus facilitates the polymerase exchange with additional deoxycytidyl transferase activity that is involved in insertion of dCMP

All Fanconi Anemia patients usually indicate hypersensitivity to cross-linking agents, signifying that FA pathway plays an indispensable role in distinguishing, beckoning or repair of lesions generated by agents. However, the precise role of FA proteins in response to ICLs is still in its infancy. FA pathway tends to participate in both replication-dependent and independent pathways of ICL repair. After an exposure of FA cells withy cross-linking agents, they accumulate chromosomal breaks and radial chromosomes [84] which is an outcome of defects in cellular responses to ICLs. After recognition of ICL and signaling cell cycle arrest, FA pathways function to coordinate the repair of ICL. Approximately, thirteen Fanconi anemia proteins are essential for resistance against ICLs and the clampdown of chromosomal stability. Eight FA proteins tend to form a nuclear protein complex in order to monoubiquitylate FancD2 and FancI. This event is crucial for the cellular resistance to ICL agents. Disruption in FA core complex and ID complex tend to decrease ICL repair efficiency [85]. The depletion of FANCD2 prevents identification of post-incision product i.e. double-strand breaks (DSB). The programmed DSB that is promoted by FANCI-FANCD2 complex majorly leads to the formation of Rad51 filaments and thus allows subsequent repair via Homologous recombination. Notably, FA pathway has been associated with proteins involved in HDR, TLS and Nucleotide excision repair. However, the exact role of FA proteins in HDR provides a vague notion. Though, there exists an interaction between the conduits of FA-BRCA, as FANCD1 exhibits homology with BRCA2 and for this reason, numerous proteins of FA pathway unswervingly interact with BRCA1 and BRCA2. In this way, it is believed that FA pathway donot play a significant role in all Homology Directed repair mechanisms (HDR), because of having a role in the recruitment of repair proteins in ICL damage. Certainly, *in vitro* analysis recommend that FANCD1/BRCA2 play a momentous role in ICL repair [86]. FANCD2 allies with the Mre11-Rad50-Nbs1 (MRN) complex, that is considerably crucial for incision of DNA strands during double-strand breaks

(DSBs), a preliminary step of all homology dependent processes [87].

*DOI: http://dx.doi.org/10.5772/intechopen.97551*

are not sensitive for cross-linking agents [82].

stranded DNA around ICL.

residues opposite to ICLs.

**8.3 FA proteins and ICL repair**

of N2 -N2 *Interstrand Crosslink Repair: New Horizons of DNA Damage Repair DOI: http://dx.doi.org/10.5772/intechopen.97551*

*DNA - Damages and Repair Mechanisms*

**8.1 Role of homologous recombination in ICL repair**

The phenomenon of homologous recombination repair (HRR) employs homolo-

gous DNA sequences as template for repair and tolerance of DNA lesions that obstruct DNA replication in S-phase. Homologous recombination usually encompasses four step (i) double strand break recognition tailed by nucleolytic processing to produce 3′ single stranded ends of DNA, (ii) protein-mediated strand invasion of single-stranded DNA with homologous chromosome (iii) synthesis of DNA which regenerates degraded DNA using undamaged homologous chromosome as a template and (iv) resolution of Holliday junction intermediates. Usually the platinum drugs drive fruitful results in the treatment of BRCA1- and BRCA2- associated ovarian cancers [76]. However, the protein products of these two genes give rise to HR-mediated repair of DNA damage. A dynamic combination of BRCA1 and associated RING domain protein 1 (BARD1) exhibits ubiquitin ligase activity that is essential for the proper localization of RAD51, which is a central player in Homologous Recombination repair. Through BRCA2 mediated interaction with RAD51, it is specifically targeted to sites where recombination is initiated [77]. However, RAD51-

deficient cells represent hypersensitivity towards ICL-inducing agents.

In this regard, the model organism, *Escherichia coli* has provided deep insights in the mechanisms involved in HRR of bacteria. Usually, RecA of bacteria has proven to be an effective protein in all major aspects of HRR due to its ability of forming nucleoprotein filament with both single and double stranded DNA. In *E. coli,* RecBCD complex- combination of nuclease/helicase, initiates the phenomenon of recombination by creating 3′-terminal single-stranded DNA substrate for the activity of RecA protein. RecBCD complex usually binds to the end of linear double stranded DNA and RecA in combination with single-stranded binding proteins (SSBP) allows an incessant formation of presynaptic filament on DNA. This nucleoprotein complex allows a rapid and efficient search for homology within the double-stranded DNA recipient, with subsequent formation of a joint molecule. After the formation of joint molecule, DNA PolI regenerates the sequence and the resultant Holliday junction is resolved by the action of RuvC protein that acts in concert with RuvAB proteins to coordinate the steps of branch migration and Holliday junction resolution [78].

In *Saccharomyces cerevisiae*, the incision of DNA is carried out by an anonymous nuclease. A yeast homologue of RecA, Rad51 works in conjunction with Rad52 dislocates the single-stranded DNA that is ostensibly covered by RPA. The subsequent nucleofilament works with Rad54 and Rad55/57 in DNA unwinding and strand annealing between donor DNA and incoming Rad51 nucleoprotein. The resolution of subsequent recombination intermediates is frequently carried out by assorted set

Translesion DNA Synthesis polymerases are considered essential for ICL repair in both S/G2 and G1 to bypass an ICL unhooked from one of the two cross-linked strands. The phenomenon of Translesion synthesis encompasses multiple polymerases with a dynamic ability to carry out an insertion of nucleotide across the lesion and others carrying out further extension. Based on genetic and biochemical studies, an assortment of polymerases has been implied in repair of ICLs. Usually translesion synthesis is a threefold step: (i) release of replicative polymerase after an interruption of normal bidirectional DNA with lesion, (ii) release of specialized translesion polymerase onto a site and starts the replication at a short distance past the lesion, (iii) the replacement of translesion polymerase with replicative DNA

of mechanisms including mus81-mms4 nuclease and Resolvase A [79].

**8.2 Translesion DNA synthesis in DNA interstrand crosslinks**

polymerase which continues the normal process of replication [80].

**130**

For HR-mediated repair of replication-dependent DSB and excision of ICL from the genome, this is vital to generate an intact template. In this regard, an assortment of polymerases allows the bypass of unhooked ICLs in vitro by using cross-linked DNA substrate model. In *Escherichia coli,* PolIV can easily bypass the unhooked ICLs of N2 -N2 -guanine in a non-mutagenic manner [81]. A set of human TLS polymerases entail Pol η, Pol ι, Pol κ, REV1, and Pol ν that tend to insert the complementary bases or evade anatomically varied ICLs. Competencies of such polymerase-catalyzed reactions is contingent upon the structure of ICL and the amount of doublestranded DNA around ICL.

The role of TLS polymerases in ICL repair is strongly supported by the study of genetics. In yeast, mutations in genes encoding subunits of Polζ i.e. Rev3, Rev7 or REV1 render cells hypersensitive to cross-linking agents [72]. Polζ is majorly important for the cross-linking resistance of non-replicating cells. However, to date *in vitro* studies have not been able to show bypass of ICL damage by Pol ζ-REV1, thus suggesting the other factors involved in lesion bypass. However, Pol η mutants are not sensitive for cross-linking agents [82].

In mammals, Pol ζ (comprising of REV3 and REV7 subunits) and REV1 are significant factors in ICL repair. However, the cells deficient in any of the aforementioned genes are highly sensitive to various cross linking agents [83]. REV1 act as TLS polymerase scaffold and thus facilitates the polymerase exchange with additional deoxycytidyl transferase activity that is involved in insertion of dCMP residues opposite to ICLs.

#### **8.3 FA proteins and ICL repair**

All Fanconi Anemia patients usually indicate hypersensitivity to cross-linking agents, signifying that FA pathway plays an indispensable role in distinguishing, beckoning or repair of lesions generated by agents. However, the precise role of FA proteins in response to ICLs is still in its infancy. FA pathway tends to participate in both replication-dependent and independent pathways of ICL repair. After an exposure of FA cells withy cross-linking agents, they accumulate chromosomal breaks and radial chromosomes [84] which is an outcome of defects in cellular responses to ICLs.

After recognition of ICL and signaling cell cycle arrest, FA pathways function to coordinate the repair of ICL. Approximately, thirteen Fanconi anemia proteins are essential for resistance against ICLs and the clampdown of chromosomal stability. Eight FA proteins tend to form a nuclear protein complex in order to monoubiquitylate FancD2 and FancI. This event is crucial for the cellular resistance to ICL agents. Disruption in FA core complex and ID complex tend to decrease ICL repair efficiency [85]. The depletion of FANCD2 prevents identification of post-incision product i.e. double-strand breaks (DSB). The programmed DSB that is promoted by FANCI-FANCD2 complex majorly leads to the formation of Rad51 filaments and thus allows subsequent repair via Homologous recombination. Notably, FA pathway has been associated with proteins involved in HDR, TLS and Nucleotide excision repair. However, the exact role of FA proteins in HDR provides a vague notion. Though, there exists an interaction between the conduits of FA-BRCA, as FANCD1 exhibits homology with BRCA2 and for this reason, numerous proteins of FA pathway unswervingly interact with BRCA1 and BRCA2. In this way, it is believed that FA pathway donot play a significant role in all Homology Directed repair mechanisms (HDR), because of having a role in the recruitment of repair proteins in ICL damage. Certainly, *in vitro* analysis recommend that FANCD1/BRCA2 play a momentous role in ICL repair [86]. FANCD2 allies with the Mre11-Rad50-Nbs1 (MRN) complex, that is considerably crucial for incision of DNA strands during double-strand breaks (DSBs), a preliminary step of all homology dependent processes [87].

In response to cross-linking agents, FANCD2 has been exposed to co-localize with Nucleotide Excision Repair component, XPF that affects the solidity of ubiquitylated FANCD2. After replication arrest, FANCD2 has also been shown to co-localize with Rev1 [88]and core complex components of FA i.e. FANCA and FANCG have been shown to be required for Rev1 foci formation [89]. Because of a dynamic ability to play an indecisive role in HDR and upstream process of TLS and NER, FA pathway orchestrates and regulate such repair mechanisms for a suitable removal of ICL damage. In this way, inactivation of FANCD2 affect both nucleolytic incision and translesion synthesis [90]. Recent investigations have examined the role of FA pathway in ICL repair by means of DNA substrates carrying site-specific ICLs in the supernatants of Xenopus.

Having a DNA substrate containing MMC-like ICL adducts significantly distorts DNA helix. The other study has stated that ICL repair can proceed through replication dependent and independent mechanisms [85]. In nutshell, ICL repair could take place in an absence of DNA replication in *Xenopus* extracts and upon transfection of an ICL- containing plasmid in G1-arrested mammalian cells is consistent with accumulating evidence for ICL repair in G1.

## *8.3.1 RUNX poly(ADP-ribosyl)ation and BLM interaction facilitate the Fanconi anemia pathway of DNA repair*

Fanconi anemia is considered as a universal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICL). The tumor suppressors RUNX1 and RUNX3 have been shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage dependent chromatin association of FANCD2. RUNX3 usually modifies by PARP-dependent poly(ADP-ribosyl) ation which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. After DNA gets damage, the increased interaction between RUNX3 and BLM facilitates the efficient FANCD2 chromatin localization. The mutations of RUNX-Walker motif in breast cancers have been impaired for DNA damage-inducible PARylation, thus unveiling an impending mechanism for FA pathway inactivation in cancers [91].

#### **8.4 Suppression of NHEJ reduces ICL sensitivity**

Even though Homologous Recombination promotes repair of double strand break in S-phase, an alternative mechanism, Non-homologous end joining (NHEJ) also exist to repair damaged DNA in all phases of the cell cycle. The phenomenon of NHEJ employs a simplest mechanism of splicing to rejoin the free end of DNA. The process involves the binding of KU70-KU80 heterodimers to the free doublestranded ends of DNA, thus allows the binding of DNA-dependent kinase subunit (DNA-PKcs) and initiates the activation of downstream steps [92]. DNA is processed to remove 5′-or 3'-ssDNA tails and the subsequent ends are directly rejoined by the activity of DNA ligase IV-XRCC4. Unlike HRR, in which homologous sequences proofread the repair process, NHEJ generates deletions, insertions and translocations in case of joining of incorrect ends.

In past, researches on mice and yeast has stated the notion that human cell lines defective in factors of Non-homologous end joining i.e. KU70, KU80, Ligase, DNA-PKcs or XRCC4, donot exhibit hypersensitivity towards ICL-inducing agents [93]. However, recent analysis has indicated that inhibition of NHEJ pathway in cell lines of FA patients can reduce the toxicity of ICL-inducing agents. For instance, in a knockout model of chicken or nematode, specific FA-like defects can be salvaged by the co-deletion of ligase IV or KU70. Moreover, through simultaneous inhibition

**133**

**Author details**

**9. Conclusion**

and Abdul Hannan2

, Javaria Zafar1

provided the original work is properly cited.

\*, Naureen Ehsan1

3 Institute of Business Administration, University of Punjab, Lahore, Pakistan

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Institute of Industrial Biotechnology, GCU, Lahore, Pakistan

\*Address all correspondence to: javariazafar614@gmail.com

2 Business School, University of Central Punjab, Lahore, Pakistan

before we get a vivid picture of this important repair mechanism.

, Qurat-Ul-Ain2

, Mahnoor Tariq3

Amna Aqeel1

*Interstrand Crosslink Repair: New Horizons of DNA Damage Repair*

Strand breaks amid non-homologous chromosomes.

of NHEJ by PKcs inhibitor, NU7036 in FANCA- and FANCD2- deficient human cell lines, the high sensitivity to MMC can be rescued easily. Through analysis of mitotic spreads in these cell lines, a rare sight of uncharacteristic radial chromosomes was observed. These annotations direct that a key purpose of the FA conduit in repair of Interstrand crosslink lesions, is to subdue the forged ligation of ICL-induced Double

HR and NHEJ pathway provides the complementary functions in the repair of *de novo* double strand breaks and the co-inhibition of these repair pathways leads to increased cell death [94]. However, Fanconi Anemia cells are not defective in HR per se, so the inhibition of NHEJ in FA cells still allows them to proliferate and repair double strand breaks. This is mainly due to the reason that FA pathway mainly endorses HR at stalled replication forks through stabilization of intermediate that is a prerequisite for unhooking and TLS. If still the replication fork is not stabilized, HR can befall but the generated free end of DNA likes to bound by KU70-KU80, as it has a very high affinity for the structures [95]. By inhibition of NHEJ pathway, the less active and less toxic FA-independent HR pathway can re-establish the replication fork.

The development of interstrand cross-links play a chief role in the mechanism of significant chemotherapeutic agents. Emerging evidences suggest that these ICL lesions may also be formed by environmental agents and unwanted byproducts of metabolic processes. A better understanding of these lesions could lead to the improvement of supplementary therapeutic agents and strategies. However, despite the efforts of considerable investigations, the mechanism of ICL repair is still an enigma. At the transcriptomic level, proteins involved in a number of repair pathways have been identified. However, the detailed analysis of conditions in which repair should occur is largely elusive. What's clear is that a repair of interstrandcross links in eukaryotes involves multiple factors from NER and HRR pathways. Given the state of activities, it is ostensible that diverse experiments need to be done

*DOI: http://dx.doi.org/10.5772/intechopen.97551*

#### *Interstrand Crosslink Repair: New Horizons of DNA Damage Repair DOI: http://dx.doi.org/10.5772/intechopen.97551*

of NHEJ by PKcs inhibitor, NU7036 in FANCA- and FANCD2- deficient human cell lines, the high sensitivity to MMC can be rescued easily. Through analysis of mitotic spreads in these cell lines, a rare sight of uncharacteristic radial chromosomes was observed. These annotations direct that a key purpose of the FA conduit in repair of Interstrand crosslink lesions, is to subdue the forged ligation of ICL-induced Double Strand breaks amid non-homologous chromosomes.

HR and NHEJ pathway provides the complementary functions in the repair of *de novo* double strand breaks and the co-inhibition of these repair pathways leads to increased cell death [94]. However, Fanconi Anemia cells are not defective in HR per se, so the inhibition of NHEJ in FA cells still allows them to proliferate and repair double strand breaks. This is mainly due to the reason that FA pathway mainly endorses HR at stalled replication forks through stabilization of intermediate that is a prerequisite for unhooking and TLS. If still the replication fork is not stabilized, HR can befall but the generated free end of DNA likes to bound by KU70-KU80, as it has a very high affinity for the structures [95]. By inhibition of NHEJ pathway, the less active and less toxic FA-independent HR pathway can re-establish the replication fork.
