**3. Chromatin modifications in DSB repair**

DSB repair can activate two repair pathways in mammalian cells: the HRR and NHEJ. First it is active throughout the cell cycle, and second it is active in S and G2 phases [36]. Usually, NHEJ is initiated by DSB recruitment of Ku70/80 or XRCC6 XRCC5 heterodimer. Ku70/80 is DBS sensor and facilitates the downstream factors recruitment, including DNA-PKcs, PAXX, XLF, XRCC4, and ligase IV. Recruitment of downstream factors helps complete DNA repair. However, HRR involves MRE11- RAD50-NBS1 (MRN) complex to activate ATM protein kinase [37]. Also, HRR can be influenced by PKcs and act in concert with the MRN complex and the recruitment of this complex to DNA DSB by hSSB1 (single-stranded binding protein), as well as the activities of 53BP1/RIF1 and BRCA1/CtIP [38]. Cells start a cascade of phosphorylation events in response to DNA damage, mediated by three phosphatidylinositol-3-related kinases: ATM, ATR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The kinases activate a rapid reaction of abundant sensors for DSBs to check injuries, recruit effectors, and generate a coordinated response to maintain the genome integrity [37, 39].

Different epigenetic modifications are related to these kinases, while H2AX is an important chromatin-based substrate for phosphatidylinositol-3-related kinases, when it is phosphorylated on S139, and is named γ-H2AX. This phosphorylated form serves as a checkpoint for HR and NHEJ. The foci formation of γH2A.X is the most often observed epigenetic modification triggered by DNA damage; together with the response to DSB and the SWI/SNF complex, it promotes phosphorylation of S139 in its C-terminal region through ATM in human cells. Domains of γ-H2AX are established by contact with the DSB site. In fact, the break site defines the densities and spread of γ-H2AX [40–42]. In addition, PBAF functions in the ATM pathway silence transcription in *cis* on DSB by promoting mono-ubiquitination of H2A on K119 [43] and efficient early repair on exposure to ionizing radiation (IR), mediated by DSB and NHEJ [44]. H2AX induction by UV depends on ATR, but the formation of DSB in late stages contributes to ATM activation and the increase of H2AX. DNA-PKcs, after induction of DSB, is responsible for H2AX and chromatin remodeling factor (KAP1) phosphorylation. Furthermore, DNA-PKcs is required for chromatin remodeling in early postirradiation stages and promotes the rapid recruitment of DDR initiation proteins at DSBs sites [37]. An early modulator is BRIT, a chromatin-binding protein that forms irradiation-induced nuclear foci (IRIF) and works as a proximal factor at checkpoints in DNA, controls multiple damage sensors, and early mediators to DDR. BRIT1 colocalizes with γ-H2AX, using ATM/ATR to form the BRIT1-SWI/SNF interaction through its BAF170 and BAF155 subunits, potentiating the response to damage [45, 46].

Moreover, γ-H2AX recruits MDC in the chromatin, is phosphorylated by ATM, functioning as the protein coupling site of checkpoints and repair, such as 53BP1 and the UBC13-RNF8-BRCA1 complex. The protein 53BP1 is phosphorylated by ATM and helps in the BRCA1 recruitment: together with the post-translational modifications in the damage site, they help promote other repair proteins. ATM and RNF8 facilitate the repair of DNA DSBs in the phases G1 and G2 of the cell cycle, regardless of the repair pathway used [47, 48]. Similarly, ubiquitination in the DSB regulates the repair protein BRCA1 and P53, as well as BARD1 recruitment by 53BP1 [49]. BRCA1 and 53BP1 are necessary to regulate downstream histones phosphorylation and ubiquitination to direct the repair on NHEJ and HR. Thus, BRCA1 repairs DNA damage via HR, and 53BP1 plays a crucial role in the NHEJ repair pathway, ensuring DNA DSBs are repaired correctly [50, 51]. BRIT1 is required for DNA damage-induced intra-S and G2/M checkpoints, as regulation of the BRCA1 and Chk1 expression [52]. The loss of function of either of these two genes can alter

the HRR pathway, resulting in genetic instability and an increased risk of breast or ovarian cancer in BRCA1/2 germline mutation carriers [53, 54]. Cruz et al. [55] analyzed the RAD51 foci in 20 samples from patients with breast cancer, 10 in germinal-BRCA1, and 10 in germinal-BRCA2. The results provide new evidence of HRR restoration functionality as a frequent mechanism of PARPi resistance and demonstrate the potential of functional biomarkers to discriminate against tumors that will fail PARPi monotherapy. This emphasizes the major role played by ATM and DBSs-associated repair pathways in breast cancer treatment, whether mediated by IR or chemotherapy.

Qi et al. [56, 57] found that BRG1 decreases the nucleosomes stability at DSBs and creates an open and relaxed chromatin structure in SW13 and U2OS cells. It shows that BRG1 is crucial in early damage repair by remodeling the chromatin structure near DNA damage sites. They show that the BRG1 domain facilitates the RPA replacement with RAD51 at the DSB site in the HR, interacting with the RAD52 mediator and regulating its recruitment into the DSBs. de Castro et al. [58] demonstrated for the first time that Arid2 expression is important for HR. They found that Baf200 and Brg1 are required for efficient recruitment of Rad51 to a subset of DSBs, repaired by HR, where Rad51 and Arid2 are part of the same complex. Haokip et al. [59] demonstrated that SMARCAL1 and BRG1 regulate each other in HeLa cells with DNA damage-inducing agent doxorubicin, resulting in an increase in the SMARCAL1 transcription and protein. They found that BRG1 is present at the Enh1 and Enh2 region of SMARCAL1 promoter in untreated HeLa cells. Experiments showed how BRG1 is present on SMARCAL1 promoter, with protein occupancy increasing when DNA is damaged, indicating that BRG1 can positively regulate SMARCAL1, creating a regulatory loop. Regarding the relationship with proteins of initial DDR pathway, Keka et al. [60] showed that the loss of Smarcal1 reduces the XRCC4 recruitment to DSB sites several times and suggests that Smarcal1 is required for DNA-PKcs/Ku70/Ku80 complex to the correct functions. Diplas et al. [61] found that loss of SMARCAL1 in glioblastoma cells can induce alternative lengthening of telomere (ALT) phenotypes, in the same way as ATRX. ATRX mutations are the most prevalent abnormality in glioma, as Han et al. [62] identified that knockout ATRX inhibited glioma cell growth, tumor invasion, and a decrease in H3K9me3 availability, which can inhibit the ATM acetylation resulting in increased glioma cell chemosensitivity. They found that ATRX is involved in DNA damage repair by regulating the ATM pathway, suggesting a good prognostic marker in predicting temozolomide (TMZ) chemosensitivity.

Although little is known about the role of H1.2 in response to damage and interaction with the repair machinery proteins, its function has become clearer. Kim et al. [63] reported that p300-mediated acetylation of p53 and DNA-PK-mediated phosphorylation of H1.2 alter the p53-H1.2 interaction, thus alleviating the repressive effects of H1.2 on the transactivation of p53. After DNA damage, p53 and H1.2 undergo modifications in an orderly fashion, with H1.2 phosphorylation at T146 followed by p53 acetylation. p53-H1.2 interaction is essential to enhance p53 function, and point mutations that mimic its constitutive modifications induce efficient growth inhibition and apoptosis. Li et al. [64] described a new mechanism to H1.2, without other H1 isoforms, to regulate the DNA damage response and repair through repression of ATM recruitment and activation. Moreover, H1.2 functions as a molecular brake for ATM binding to MRN, whereas DNA damage-induced ATM activation requires both the MRN complex assembly and H1.2 release. As such, these authors revealed a new link between chromatin disturbances, destabilization of H1.2, and ATM activation. For the first time, it was found that the RNF168/ RAD6 complex can promote mono-ubiquitination of histone H1.2 *in vitro*, and the H1.2 mono-ubiquitination can be induced after IR treatment. They concluded that

**173**

*Epigenetics and DNA Repair in Cancer DOI: http://dx.doi.org/10.5772/intechopen.94030*

agents acting independently of p53.

**4. Methylation in DNA damage and repair**

treatment, particularly by radiotherapy [72].

H1.2 mono-ubiquitination *in vitro* and *in vivo* by RNF168/RAD6 is evidence that mono-ubiquitination can establish a suitable microenvironment for other E2/E3 complexes to catalyze polyubiquitination or multi-mono-ubiquitination of H2A and H2AX over H2A and H2AX ubiquitination, which is dependent on RNF8/Ubc13 [65]. Thorslund et al. [66] proposed that linker H1 represents a key chromatinassociated RNF8 substrate, whose UBC13-dependent K63-bound ubiquitylation at DSB-containing chromatin provides a scaffold to RNF168 binding through its UDM1 module. RNF168 ubiquitinates H2A into K13/K15 and possibly other proteins to trigger repair factor recruitment in DSB. Giné et al. [67] implicated H1.2 as a valuable protein for apoptosis induction in chronic lymphocytic leukemia cells, and its release pattern is correlated with deletions of 17p and treatment response; they emphasized that histone H1.2 could be an essential apoptotic signal induced by

DNA methylation is considered a post-replication modification by the methyl group (–CH3) addition at carbon 5 cytosine, known as 5-methylcytosine (5mC), primarily in dinucleotides of CpG [68]. This enzymatic reaction is catalyzed by three DNA methyltransferases (DNMTs) (DNMT1, DNMT3A, and DNMT3B). These modifications are commonly found in promoter regions, the CpG islands, as their main function is transcription silencing, decreasing, or repressing the gene function. Methylation is the epigenetic modification most studied in cancer, as it is well-known that DNA hypermethylation can transcriptionally silence tumor suppressors and DNA repair genes, giving neoplastic cells survival advantages [69]. Various genes related to cell cycle regulation, tumor cell invasion, cell signaling, apoptosis, and chromatin remodeling are hypermethylated and silenced in almost all tumors. As demonstrated by Pal et al. [70] for the first time, CpG sites in the promoter of H2AX, RNF8, and CYCS are methylated; they show the collaborative participation of hypermethylation of DR5, DCR1, DCR2, CASP8, CYCS, BRCA1, BRCA2 and H2AX and the hypomethylation of DR4, FLIP, and RNF8 in sporadic breast cancer; the authors proposed that promoter methylation of these apoptotic and DDR genes is not due to a random phenomenon, as the progressive modification of aberrant epigenetic alterations are associated with tumor advancement, which generates the dysregulation of the DDR-apoptotic pathway, promoting tumor development. Hinrichsen et al. [71] found that increased methylation in the promoters of MLH1, MSH2, PMS2, and p16 genes are correlated to an advanced stage in hepatocellular carcinoma. Epigenetic marks and DDR are crucial points to understand if changes in DNA methylation can contribute to resistance of cancer

Various epigenetic marks have been associated with the response to damage, primarily the methylation of lysine 4 and 79 in H3 (H3K4me and H3K79me, respectively) that contribute to DDR and DNA repair [73]. The marks on H3K4me1/2/3 regulate the repair of DSBs through chromatin accessibility [74, 75]. Furthermore, H3K36 methylation is associated with "open" euchromatin and helps RNA pol II activate transcription [76]. Chang et al. [77] demonstrated that the tumor suppressor PHRF1 can move on the DSB due to H3K36me2/me3 and NBS1, then ubiquitinate PARP1, and trigger the subsequent repair by NHEJ. Yet, it is known that DOT1L methyltransferase catalyzes the H3K79 mono-di-trimethylation through its non-SET domain. Recently, in the study by Kari et al. [78], the depletion or inhibition of DOT1L activity was shown to result in altered DNA damage response, indicated by decreased levels of γH2AX, but with increased KAP1 phosphorylation. Loss

#### *Epigenetics and DNA Repair in Cancer DOI: http://dx.doi.org/10.5772/intechopen.94030*

*DNA - Damages and Repair Mechanisms*

by IR or chemotherapy.

predicting temozolomide (TMZ) chemosensitivity.

Although little is known about the role of H1.2 in response to damage and interaction with the repair machinery proteins, its function has become clearer. Kim et al. [63] reported that p300-mediated acetylation of p53 and DNA-PK-mediated phosphorylation of H1.2 alter the p53-H1.2 interaction, thus alleviating the repressive effects of H1.2 on the transactivation of p53. After DNA damage, p53 and H1.2 undergo modifications in an orderly fashion, with H1.2 phosphorylation at T146 followed by p53 acetylation. p53-H1.2 interaction is essential to enhance p53 function, and point mutations that mimic its constitutive modifications induce efficient growth inhibition and apoptosis. Li et al. [64] described a new mechanism to H1.2, without other H1 isoforms, to regulate the DNA damage response and repair through repression of ATM recruitment and activation. Moreover, H1.2 functions as a molecular brake for ATM binding to MRN, whereas DNA damage-induced ATM activation requires both the MRN complex assembly and H1.2 release. As such, these authors revealed a new link between chromatin disturbances, destabilization of H1.2, and ATM activation. For the first time, it was found that the RNF168/ RAD6 complex can promote mono-ubiquitination of histone H1.2 *in vitro*, and the H1.2 mono-ubiquitination can be induced after IR treatment. They concluded that

the HRR pathway, resulting in genetic instability and an increased risk of breast or ovarian cancer in BRCA1/2 germline mutation carriers [53, 54]. Cruz et al. [55] analyzed the RAD51 foci in 20 samples from patients with breast cancer, 10 in germinal-BRCA1, and 10 in germinal-BRCA2. The results provide new evidence of HRR restoration functionality as a frequent mechanism of PARPi resistance and demonstrate the potential of functional biomarkers to discriminate against tumors that will fail PARPi monotherapy. This emphasizes the major role played by ATM and DBSs-associated repair pathways in breast cancer treatment, whether mediated

Qi et al. [56, 57] found that BRG1 decreases the nucleosomes stability at DSBs and creates an open and relaxed chromatin structure in SW13 and U2OS cells. It shows that BRG1 is crucial in early damage repair by remodeling the chromatin structure near DNA damage sites. They show that the BRG1 domain facilitates the RPA replacement with RAD51 at the DSB site in the HR, interacting with the RAD52 mediator and regulating its recruitment into the DSBs. de Castro et al. [58] demonstrated for the first time that Arid2 expression is important for HR. They found that Baf200 and Brg1 are required for efficient recruitment of Rad51 to a subset of DSBs, repaired by HR, where Rad51 and Arid2 are part of the same complex. Haokip et al. [59] demonstrated that SMARCAL1 and BRG1 regulate each other in HeLa cells with DNA damage-inducing agent doxorubicin, resulting in an increase in the SMARCAL1 transcription and protein. They found that BRG1 is present at the Enh1 and Enh2 region of SMARCAL1 promoter in untreated HeLa cells. Experiments showed how BRG1 is present on SMARCAL1 promoter, with protein occupancy increasing when DNA is damaged, indicating that BRG1 can positively regulate SMARCAL1, creating a regulatory loop. Regarding the relationship with proteins of initial DDR pathway, Keka et al. [60] showed that the loss of Smarcal1 reduces the XRCC4 recruitment to DSB sites several times and suggests that Smarcal1 is required for DNA-PKcs/Ku70/Ku80 complex to the correct functions. Diplas et al. [61] found that loss of SMARCAL1 in glioblastoma cells can induce alternative lengthening of telomere (ALT) phenotypes, in the same way as ATRX. ATRX mutations are the most prevalent abnormality in glioma, as Han et al. [62] identified that knockout ATRX inhibited glioma cell growth, tumor invasion, and a decrease in H3K9me3 availability, which can inhibit the ATM acetylation resulting in increased glioma cell chemosensitivity. They found that ATRX is involved in DNA damage repair by regulating the ATM pathway, suggesting a good prognostic marker in

**172**

H1.2 mono-ubiquitination *in vitro* and *in vivo* by RNF168/RAD6 is evidence that mono-ubiquitination can establish a suitable microenvironment for other E2/E3 complexes to catalyze polyubiquitination or multi-mono-ubiquitination of H2A and H2AX over H2A and H2AX ubiquitination, which is dependent on RNF8/Ubc13 [65]. Thorslund et al. [66] proposed that linker H1 represents a key chromatinassociated RNF8 substrate, whose UBC13-dependent K63-bound ubiquitylation at DSB-containing chromatin provides a scaffold to RNF168 binding through its UDM1 module. RNF168 ubiquitinates H2A into K13/K15 and possibly other proteins to trigger repair factor recruitment in DSB. Giné et al. [67] implicated H1.2 as a valuable protein for apoptosis induction in chronic lymphocytic leukemia cells, and its release pattern is correlated with deletions of 17p and treatment response; they emphasized that histone H1.2 could be an essential apoptotic signal induced by agents acting independently of p53.
