**3. ICL Repair genes and human disorders**

The proteins involved in the repair of ICLs have vital role in pathophysiology of various hereditary disorders for example xeroderma pigmentosum (XP), cerebrooculo-facio-skeletal syndrome (COFS), Fanconi Anaemia (FA), trichothyodistrophy as well as Cockayne syndrome (CS) [37]. FA is associated with aplastic anemia, cancers (often acute myelogenous leukemia) and bone marrow failure. The mutational changes in any *FANC* genes contribute towards genomic instability and the sensitivity against the ICL agents [38]. According to an estimate 18 genes are involved in FA and the products of genes collaborate for ICL repair during the S phase [39]. Apart from these, the defective NER pathways also result in several rare autosomal-recessive diseases like XP, CS, TTD and COFS syndrome [40]. Moreover, there are 11 genes that are associated with NER pathways and the defect in these occur due to the mutations in these genes. XP is associated with pigmentation, photosensitivity as well as cancerous skin diseases. Another inherited syndrome known as CS is present in which there are several problems arises namely ocular defects, mental deficiency, extensive demyelination, short stature, photosensitivity, large hands, feet, as well as ears [37]. There are wide ranging clinical spectrum of CS and the patients acutely affected are categorized under COFS syndrome patients. TTD is associated with neuro-ectodermal symptoms and clear sulfur-deficient brittle hair [41]. These NER diseases are different from each other with respect to their physical characteristics involving cutaneous ailments.

Keeping in view the various DNA repair factors, ICL genes has found to be having a strong link with cancer. There are several genes that are revealed by next-generation sequencing and play a part in hereditary breast cancer as well as ovarian cancer syndrome (HBOC). These genes are *BRCA1, BRCA2, PALB2, BRIP1 and RAD51C* exhibiting a close link with HBOC in the ICL repair pathways [42]. The preventive medication strategy requires the early detection of the mutations happening in BRCA1 and BRCA2 genes to help in process of recovery.

## **4. Recognition of ICL lesions in mammalian cells**

During the course of ICL damage, the UHRF1 protein comes to rescue at the site within a fraction of seconds [43]. These proteins identify ICLs with the help of its SET and RING finger associated (SRA) domain, the same domain notable for its recognition ability for the hemi-methylated DNA and employment of DNMT1 to ensure the maintenance of methylation signature in the cells of mammals [44]. The relative affinity of UHRF1 protein in response to hemi-methylated DNA as well as ICLs are somewhat similar and proposed that UHRF1 interacted with both of them through related mechanisms. The UHRF1 proteins are employed preceding the incorporation of FANCD2 to ICLs [43]. About 10 minutes are lagged between the assembling of UHRF1 and FANCD2 to ICLs. This strengthens the assumption of other proteins being employed or the other PTM events that might occur during this time interval. The proper mechanism of UHRF1 mediated FANCD2 repair is not clear but implicate a direct protein–protein interaction. There has also been a proposed role of UHRF1 in a nuclease scaffold [45]. It is also proposed that the rapid incorporation of UHRF1 to the ICLs paves the way for FA mediated repair of lesion later on. As ICLs vary in their structural framework, there is a probability that in addition to UHRF1, other ICL sensor proteins do exist in the same way.

### **5. Factors involved in ICL repair pathway**

There are several proteins that take part in the ICL repair. Along with these, included 15 proteins that are not only specific to FA genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, and P) but also to other repair pathways [46]. The important recombination factors like RAD51, the structure-specific endonucleases like MUS81/EME1 and XPF/ERCC1, translesion DNA polymerases and Holliday junction processing factors all contribute towards the repair of ICLs.

A rare human genetic disease known as FA, which is associated with pancytopenia, various developmental abnormalities and a high cancer risk [47]. The cells procured from FA patients depict the large amount of chromosomal breakage as well as the formation of radial chromosomes [48] that bring strength to the idea of

**127**

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

high genomic stability in the ICL repair-deficient cells. The classical FA pathway has FA core complex (consisting of A, G, FAAP20, C, E, F, B, L, and FAAP100), an E3 ubiquitin ligase activity and the catalytic activity dedicated to the RING domain comprising FANCL protein. The core complex also acts on monoubiquitination of FANC1/D2 complex and is stimulated by damaged DNA [49]. The next step is the utilization of other downstream effectors that are attracted by the activated complex. These comprises nucleases, homologous recombination factors and translesion polymerases to remediate the lesions [50]. Whereas the exact function of monou-

An ATP dependent DEAH domain helicase namely FANCM exhibit a DNA translocase activity. It combines with FAAP24 and forms a complex structure comprising a histone-fold complex i-e MHF1/MHF2. It is a significant part of activated FA pathway [51]. The biochemical analysis also proposed that FANCM/FAAP24 complex is responsible for stabilizing and remodeling the stopped replication forks of DNA [52]. The complex of FAAP24 plays a vital part in the checkpoint activation that also need ATR to begin its function [53]. However, FANCM takes part in recombination independent ICL remediation by stimulating ubiquitination of PCNA thus promotes

The group of genes associated with FA comprises of FANCD1 (BRCA2), FANCJ, FANCN, as well as FANCO are the recombination factors that forms a connection with susceptibility for breast or ovarian cancer. The downstream processing of ICL require the employment of recombination factors, mostly when there are the double strand breaks in the DNA. The paralogous gene of FANCO (RAD51C) is RAD51 [54]. FANCO forms complex structures on interaction with RAD51B, RAD51D, XRCC2, as well as XRCC3. Another significance of these paralogs is the utilization of the recombinase RAD51 while managing a single stranded DNA [55]. RAD51 and its paralogs are vital to cells tolerant against ICLs and vice versa because they provide the homologous recombination in response to ICLs as well as the double strand breaks [56]. The endonucleases also pay a part in ICLs repair. Three important heterodimeric structure-specific endonucleases are MUS81/EME1, SLX1/SLX4 and XPF/ERCC1. SLX4 is often mutated in the complementation group consisting of FANCP [57]. The combination of SLX4 and SLX1 make up a heterodimeric nuclease. Its function is to resolve the Holliday junction formed during the remediation of ICls [58]. During the process, SLX4 act as a scaffold protein that combines the multi-activity nuclease complex comprising MUS81/EME1 as well as XPF/ERCC1. The latter acts in either of the NER pathway as well as ICL repair. The studies proposed that NER works independent of SLX4 with XPF/ERCC1 complex and the analysis of FANCP patients further strengthens the idea as they were resistant against the UV radiations [59]. Further studies suggest that XPF/ERCC1 activity requiring SLX4 involves the complete detaching in ICL repair. It is a replication dependent remediation of ICLs [60]. Digesting nuclease (SNM1A) then follows and digest the detached oligonucleotides [61]. This step is a better alternative as compared to the bypass step used for synthesis. Moreover, the lately discovered nuclease FAN1 also has a significant part in remediation of ICL. The ubiquinated FANCD2 aids in employing FAN to ICL regions. This step is mediated with the ubiquitin-binding zinc finger domain that is present in FAN1 [62]. Another important domain of FAN1 exhibit 5′-3′ exonuclease activity as well as structure-specific endonuclease activity at 5′ [63]. FAN1 thus cuts the exposed

the incorporation of other NER incision factors to the sites with ICLs [51].

ends of DNA along with DNA replication structures that hinders the process.

Other important participants in ICL repair are the translesion DNA polymerases. The blockage of normal replicative DNA polymerases is done before reaching the ICL regions. Other translesion polymerases in *Xenopus laevis* include Y-family polymerase Rev1 as well as B-family polymerase Pol ζ (Rev3/Rev7) have a significant part in complete removal of ICLs. These models also use replisome remodeling

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

biquinated FANCD2 is still ambiguous.

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

*DNA - Damages and Repair Mechanisms*

characteristics involving cutaneous ailments.

autosomal-recessive diseases like XP, CS, TTD and COFS syndrome [40]. Moreover, there are 11 genes that are associated with NER pathways and the defect in these occur due to the mutations in these genes. XP is associated with pigmentation, photosensitivity as well as cancerous skin diseases. Another inherited syndrome known as CS is present in which there are several problems arises namely ocular defects, mental deficiency, extensive demyelination, short stature, photosensitivity, large hands, feet, as well as ears [37]. There are wide ranging clinical spectrum of CS and the patients acutely affected are categorized under COFS syndrome patients. TTD is associated with neuro-ectodermal symptoms and clear sulfur-deficient brittle hair [41]. These NER diseases are different from each other with respect to their physical

Keeping in view the various DNA repair factors, ICL genes has found to be having a strong link with cancer. There are several genes that are revealed by next-generation sequencing and play a part in hereditary breast cancer as well as ovarian cancer syndrome (HBOC). These genes are *BRCA1, BRCA2, PALB2, BRIP1 and RAD51C* exhibiting a close link with HBOC in the ICL repair pathways [42]. The preventive medication strategy requires the early detection of the mutations

During the course of ICL damage, the UHRF1 protein comes to rescue at the site within a fraction of seconds [43]. These proteins identify ICLs with the help of its SET and RING finger associated (SRA) domain, the same domain notable for its recognition ability for the hemi-methylated DNA and employment of DNMT1 to ensure the maintenance of methylation signature in the cells of mammals [44]. The relative affinity of UHRF1 protein in response to hemi-methylated DNA as well as ICLs are somewhat similar and proposed that UHRF1 interacted with both of them through related mechanisms. The UHRF1 proteins are employed preceding the incorporation of FANCD2 to ICLs [43]. About 10 minutes are lagged between the assembling of UHRF1 and FANCD2 to ICLs. This strengthens the assumption of other proteins being employed or the other PTM events that might occur during this time interval. The proper mechanism of UHRF1 mediated FANCD2 repair is not clear but implicate a direct protein–protein interaction. There has also been a proposed role of UHRF1 in a nuclease scaffold [45]. It is also proposed that the rapid incorporation of UHRF1 to the ICLs paves the way for FA mediated repair of lesion later on. As ICLs vary in their structural framework, there is a probability that in

happening in BRCA1 and BRCA2 genes to help in process of recovery.

addition to UHRF1, other ICL sensor proteins do exist in the same way.

tion processing factors all contribute towards the repair of ICLs.

There are several proteins that take part in the ICL repair. Along with these, included 15 proteins that are not only specific to FA genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, and P) but also to other repair pathways [46]. The important recombination factors like RAD51, the structure-specific endonucleases like MUS81/EME1 and XPF/ERCC1, translesion DNA polymerases and Holliday junc-

A rare human genetic disease known as FA, which is associated with pancytopenia, various developmental abnormalities and a high cancer risk [47]. The cells procured from FA patients depict the large amount of chromosomal breakage as well as the formation of radial chromosomes [48] that bring strength to the idea of

**5. Factors involved in ICL repair pathway**

**4. Recognition of ICL lesions in mammalian cells**

**126**

high genomic stability in the ICL repair-deficient cells. The classical FA pathway has FA core complex (consisting of A, G, FAAP20, C, E, F, B, L, and FAAP100), an E3 ubiquitin ligase activity and the catalytic activity dedicated to the RING domain comprising FANCL protein. The core complex also acts on monoubiquitination of FANC1/D2 complex and is stimulated by damaged DNA [49]. The next step is the utilization of other downstream effectors that are attracted by the activated complex. These comprises nucleases, homologous recombination factors and translesion polymerases to remediate the lesions [50]. Whereas the exact function of monoubiquinated FANCD2 is still ambiguous.

An ATP dependent DEAH domain helicase namely FANCM exhibit a DNA translocase activity. It combines with FAAP24 and forms a complex structure comprising a histone-fold complex i-e MHF1/MHF2. It is a significant part of activated FA pathway [51]. The biochemical analysis also proposed that FANCM/FAAP24 complex is responsible for stabilizing and remodeling the stopped replication forks of DNA [52]. The complex of FAAP24 plays a vital part in the checkpoint activation that also need ATR to begin its function [53]. However, FANCM takes part in recombination independent ICL remediation by stimulating ubiquitination of PCNA thus promotes the incorporation of other NER incision factors to the sites with ICLs [51].

The group of genes associated with FA comprises of FANCD1 (BRCA2), FANCJ, FANCN, as well as FANCO are the recombination factors that forms a connection with susceptibility for breast or ovarian cancer. The downstream processing of ICL require the employment of recombination factors, mostly when there are the double strand breaks in the DNA. The paralogous gene of FANCO (RAD51C) is RAD51 [54]. FANCO forms complex structures on interaction with RAD51B, RAD51D, XRCC2, as well as XRCC3. Another significance of these paralogs is the utilization of the recombinase RAD51 while managing a single stranded DNA [55]. RAD51 and its paralogs are vital to cells tolerant against ICLs and vice versa because they provide the homologous recombination in response to ICLs as well as the double strand breaks [56].

The endonucleases also pay a part in ICLs repair. Three important heterodimeric structure-specific endonucleases are MUS81/EME1, SLX1/SLX4 and XPF/ERCC1. SLX4 is often mutated in the complementation group consisting of FANCP [57]. The combination of SLX4 and SLX1 make up a heterodimeric nuclease. Its function is to resolve the Holliday junction formed during the remediation of ICls [58]. During the process, SLX4 act as a scaffold protein that combines the multi-activity nuclease complex comprising MUS81/EME1 as well as XPF/ERCC1. The latter acts in either of the NER pathway as well as ICL repair. The studies proposed that NER works independent of SLX4 with XPF/ERCC1 complex and the analysis of FANCP patients further strengthens the idea as they were resistant against the UV radiations [59]. Further studies suggest that XPF/ERCC1 activity requiring SLX4 involves the complete detaching in ICL repair. It is a replication dependent remediation of ICLs [60]. Digesting nuclease (SNM1A) then follows and digest the detached oligonucleotides [61]. This step is a better alternative as compared to the bypass step used for synthesis.

Moreover, the lately discovered nuclease FAN1 also has a significant part in remediation of ICL. The ubiquinated FANCD2 aids in employing FAN to ICL regions. This step is mediated with the ubiquitin-binding zinc finger domain that is present in FAN1 [62]. Another important domain of FAN1 exhibit 5′-3′ exonuclease activity as well as structure-specific endonuclease activity at 5′ [63]. FAN1 thus cuts the exposed ends of DNA along with DNA replication structures that hinders the process.

Other important participants in ICL repair are the translesion DNA polymerases. The blockage of normal replicative DNA polymerases is done before reaching the ICL regions. Other translesion polymerases in *Xenopus laevis* include Y-family polymerase Rev1 as well as B-family polymerase Pol ζ (Rev3/Rev7) have a significant part in complete removal of ICLs. These models also use replisome remodeling 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 succeeded by Pol ζ that extends the unpaired strand.
