**7. Does chromatin organization affect BER? Understanding BER at the chromatin context**

 BER, as studied *in vitro* with naked DNA substrates, involves sequential enzymatic steps in which each enzyme utilizes the product of the previous step as the substrate. This observation inspired the prevailing dogma that the sequential steps in BER involves the hand-off process where the product of one step is handed over to the enzyme in the next step [153, 154]. Later steps generate intermediate product lesions that are more toxic than the original lesions. The BER intermediates such as AP sites and SSBs, which are highly mutagenic, interfere with replication and transcription, and hence the entire BER steps must be coordinated once the repair is initiated [155–158]. Cumulating evidence suggests that the BER proteins act in concert beyond simply recognizing and acting upon the product of the previous step, by being present at the site of the original lesion [43, 52, 61, 62, 64, 65, 125, 159, 160]. This is the basis for the emerging paradigm of "preformed BER complexes," named, "BERosomes" in mammalian cells. Being an integral part of complexes, it may be easier for the BER intermediates to be handed over to the next enzyme, which likely undergoes allosteric changes after binding to its substrate. Recent studies in our and collaborators' labs suggest that these "BERosomes" are constitutively chromatin-bound to ensure prompt repair in the event of any threat [62, 135, 161]. Simultaneously, recent interests in the BER field have evolved toward deciphering the role of different chromatin factors and the underlying chromatin remodeling in oxidized base repair.

#### *Regulation of Oxidized Base Repair in Human Chromatin by Posttranslational Modification DOI: http://dx.doi.org/10.5772/intechopen.81979*

 Several *in vitro* studies showed reduced BER activity with reconstituted core nucleosome particles, where every step during repair of diverse lesions was found to be inhibited by histones [162–170]. Overall BER efficiency is strongly inhibited by the presence of nucleosomes, which interfere with the interaction between the repair proteins and their substrate lesions, thereby compromising physical interaction and catalysis. Because oxidized bases perturb the DNA structure only mildly [170], whether chromatin remodeling occurs during BER was questionable. But, as BER efficiently occurs in cells, the results from these *in vitro* experiments imply that chromatin rearrangement occurs at oxidized DNA damage sites *in cells*, as was shown in the case of repair of DSBs, UV ray-mediated damages, and mismatched base pairs [171–173].

An inverse correlation exists in cells between BER and chromatin compaction. ROS induces assembly of BER complexes preferentially on open chromatin regions [174], as we have also observed that the BER complexes are constitutively present on actively transcribing sequences [175]. Interestingly, BER is involved during active CpG demethylation in promoters, mediated by TET dioxygenase(s) during transcriptional activation [176–180]. The TET proteins oxidize 5mC to 5hmC, 5fC, and 5caC; 5fC and 5caC are the TDG substrates. Thus, this coordination between CpG DNA demethylation, an epigenetic process essential for chromatin decondensation during transcriptional activation, and base damage repair supports our notion that "open-chromatin prefers BER activity across the genomic landscape" and highlights a regulatory link between epigenetics, chromatin remodeling, and BER.

 Various ATP-dependent chromatin remodeling (ACR) complexes, which play significant roles in protein/DNA and protein/protein interactions in chromatin and regulate transcription, DNA repair processes such as DSB repair (DSBR), nucleotide excision repair (NER), and cross-link repair, also affect BER. ACR complexes utilize the energy of ATP hydrolysis to restructure nucleosomes on chromatin [181–183], thereby affecting gene expression profile and DNA repair. Four structurally related, but functionally distinct, ACR complex families were identified: SWI/SNF (switching defective/sucrose nonfermenting; most extensively studied), ISWI (imitation switch), CHD (chromodomain, helicase, DNA binding), and INO80 (inositol requiring 80). *In vitro* BER studies with reconstituted nucleosomes showed enhanced repair activity in the presence of purified SWI/SNF or ISW1/ISW2 complexes [184–186]. There are some indirect evidences of ACR during BER in yeast and mammalian cells. Depletion of STH1 (ATPase subunit of RSC, a member of SWI/SNF family) causes genome-wide BER inhibition and thus emphasizes a link between chromatin organization and BER [187]. In a recent study, depletion of ALC1/CHD1L, another member of SWI/SNF, compromises chromatin relaxation, associated with BER inhibition and increased sensitivity to MMS and H2O2 in chicken cells [188]. On the contrary, INO80 deficiency in MMS-sensitive yeast cells has no effect on genome-wide BER [189]. K56 acetylation in histone H3 is increased in chromatin of both yeast and mammalian cells following MMS treatment, which generates alkylated base substrates for BER, [190, 191]. H3K56Ac was also found to be enriched at DSBR sites and responsible for SWI/SNF complex recruitment during transcription [192]. Thus, it would be interesting to examine if any specific PTM(s) would target ACR after oxidized base damage and illuminate the phenomenon of ACR during BER. In any event, additional studies are required to test if ACR plays a role in enabling BER in condensed chromatin. It would be also of interest to explore if the BER proteins possess inherent chromatin remodeling activities, similar to the NER proteins, which have SWI/SNF domains [193–195]. Though no known BER proteins have SWI/SNF domains, the XRCC1-Lig IIIα complex could disrupt nucleosomes *in vitro* and enable BER completion [166].

Poly-ADP-ribosylation of histones by PARP1 after genome damage adds negative charge on histones and disrupts histone-DNA interactions, thereby promoting chromatin decondensation and enhancing interaction between the proteins involved in DNA transactions and DNA [111, 196–198]. This could increase DNA accessibility to the BER proteins. Although PARP1's role in regulating transcription is well established, this would link chromatin remodeling to BER.

Nucleosomes pose obstruction to all DNA transactions and are likely disassembled to allow DNA replication, repair, and transcription, followed by their reassembly, which utilizes both parental histones and newly synthesized histones. Such replication-coupled nucleosome assembly in the S phase or replication-independent, transcription-coupled assembly throughout the cell cycle involves histone chaperones functioning at multiple steps of nucleosome formation [172, 199, 200]. Replicationcoupled nucleosome assembly is aided by the chromatin assembly factor (CAF-1) and Rtt106 with the help of antisilencing function 1A (ASF1A) protein. Histone cell cycle regulator (HIRA) protein, along with Daxx, mediates replication-independent nucleosome assembly. While exploring chromatin-bound BER complexes, we serendipitously discovered CHAF1A (the largest subunit of CAF-1, along with other subunits CHAF1B and RBBP4), ASF1A, and various H3/H4 variants in the immunoprecipitation complex of NEIL1 or acetylated NEIL1 (201; unpublished). This underscores the importance of the diverse chromatin components in preformed "BERosomes," which could regulate oxidize base repair in chromatin. We showed that ROS-induced oxidized base lesions caused transient dissociation of CHAF1A, ASF1A, and histones from the BER complexes and were restored back after repair completion. The repair activities of NEIL1 and OGG1, as well as complete cellular BER, were found to be inhibited by CAF-1, as well as the CHAF1A monomer [201]. So, we propose a hypothesis of temporal regulation of BER by the histone chaperones, whose dissociation from BER complexes is essential to initiate BER [201]. This has been illustrated in **Figure 2**.

Recently, we discovered acetylation of NEIL1 at the disordered C-terminal K296-K298 by p300, which enhances its activity, and found that acetylated NEIL1 (AcNEIL1) could be detected only in the chromatin fraction and not in the soluble nuclear fraction [161]. Although the nonacetylable NEIL1 3KRmutant (Lys296–298 substituted with Arg) translocates to the nucleus and binds to chromatin, presumably due to retention of positive charges as in the WT enzyme, it forms less stable BER complexes with the histones, histone chaperones, and downstream BER proteins. Thus, as proposed earlier [65], the positive charge cluster in the disordered C-terminal region is required for NEIL1's nonspecific DNA binding, after which acetylation occurs on the chromatin. Hydrophobic interaction of NEIL1 after acetylation-mediated charge neutralization probably stabilizes NEIL1's complexes with nucleosome components and downstream BER proteins. Consequently, cells with acetylable NEIL1 exhibit enhanced BER efficiency and are less sensitive to oxidative stress. It is thus likely that unmodified NEIL1 binds to chromatin nonspecifically, and acetylation specifically at the promoter regions of actively transcribing genes by enhanced p300 activity actually stabilizes NEIL1's (and possibly other DG's) BERosomes on these preferred chromatin regions (**Figure 3**), which warrants further investigation.

In a separate study, while investigating how APE1 repairs AP sites in cells, our collaborator's lab found that acetylated APE1 (AcAPE1), like AcNEIL1, is exclusively and stably chromatin-bound throughout the cell cycle [135]. APE1 undergoes acetylation after binding to AP sites in chromatin, which enhances its enzymatic activity. In the absence of APE1 acetylation, cells accumulated AP sites and exhibited higher sensitivity to DNA damaging agents. We predict that other BER proteins OGG1 and MPG, whose repair activity is enhanced by acetylation, are similarly stabilized in chromatin-bound state.

*Regulation of Oxidized Base Repair in Human Chromatin by Posttranslational Modification DOI: http://dx.doi.org/10.5772/intechopen.81979* 

#### **Figure 2.**

*A schematic showing chromatin-bound BER complexes with histones and histone chaperones. ROS-induced damage causes transient dissociation of histones and histone chaperones to initiate BER, which are restored back after repair completion.* 

**Figure 3.** 

*An illustrative view of "open" chromatin regions, containing bound "BERosomes" with histones, histone chaperones, PARPs, TETs, etc., for preferential repair of these transcriptionally active regions.* 

#### **8. Future perspectives**

The genome-wide impact of various PTMs in the cross-talks among BER proteins, which dictates the overall repair efficiency, thus preserving genomic integrity against genotoxic insults from both endogenous and external oxidative stress, has not been investigated. In this NextGen era, holistic, whole-genome scanning approaches, although a daunting challenge, make it likely to map individual PTMs of BER proteins, the kinetics of their formation and removal, and their correlation with both intrinsic and ROS-induced BER efficiency across the genomic landscape. Because histone PTMs have been well established in chromatin remodeling, it is also important to explore how specific histone PTMs interfere with the BER PTMs.

The Access-Repair-Restore model [182, 202] provides an accepted view of DNA repair in chromatin, where chromatin remodeling is essential for the DNA repair machineries to get access to the damaged DNA. For BER, it is still not clear how chromatin remodeling and the associated histone PTMs initiate BER. The BER complexes constitutively bind to"open" chromatin regions, and chromatin remodeling could assist specific enzyme-substrate binding and enzyme catalysis needed to initiate and propagate BER. Moreover, although chromatin remodeling has been found to enable BER, the enhanced repair activity may be simply due to ROS-induced stimulation of BER genes' expression or their specific PTM (acetylation), as has been shown by us [203–205], along with enhanced substrate binding in"open" chromatin. This may underestimate the contribution of ACR complexes at oxidized base lesion sites to enhance BER. Alternatively, in cells, chromatin remodeling-stimulated BER could be linked to replication and transcription, similar to transcription-coupled NER, which always occurs on"open" chromatin [206]. Indeed, repair of oxidized bases preferentially occurs in the transcribed strand [175], which could be assisted by Cockayne syndrome protein B (CSB), a NER factor, in transcription-coupled but NER-independent fashion [207]. Because BER/SSBR proteins such as PARP1 and APE1 are emerging as potential

therapeutic targets [208–212], understanding if and how chromatin remodeling impacts BER activity is crucial to manipulating BER for effective modulation of repair activity in cancer cells. This would provide better efficacy and specificity in cancer therapy.
