**5. Prereplicative BER of oxidized bases**

 The genomic integrity is particularly vulnerable during replication. Transient single-stranded (ss) DNA serving as a template during DNA replication after unwinding of the duplex genome is particularly vulnerable to ROS, which induces oxidized bases, sugar fragments, as well as strand breaks. Most oxidized bases do not stall replicative DNA polymerases, but they mispair during replication, thereby causing mutations. In contrast, bulky lesions, which stall replicative polymerases, block replisomes so as to allow repair. However, blocked replication may also lead to fork collapse, causing significant alteration in genomic stability. Furthermore, oxidized deoxynucleotides may be incorporated into the progeny strand during replication. If left unrepaired, these mutations could accumulate in progeny cells, a recipe for pathologies linked to genomic instability, including cancer, accelerated aging, and degenerative brain diseases [59, 60]. Repair of oxidative lesions, which are generated at much higher abundance than the bulky adducts in the replicating genome, is thus critical to maintain genomic fidelity. Mammalian cells have developed multiple ways to faithfully repair such base damages via prereplicative repair in the template strand and postreplicative repair in the progeny strand, immediately after replicative synthesis. Both the pathways involve an intricate collaboration of specific repair machinery with the replication proteins, likely via formation of dynamic "preformed" "repair-replication complexes" at the replication fork [61, 62].

 Repair of most mutagenic base lesions except 8-oxoG, for example, 5-OHU, TG, 5-OHC, Fapy-A, 8-oxoA, and UG must be carried out prior to replication in order to prevent mutation fixation. How such lesions, which do not block replicative Pol δ, are flagged for prereplicative repair without causing DSBs was unclear. Our recent study showed that the mammalian DG NEIL1 binds to the oxidized lesion sites in ss DNA substrates *in vitro* to facilitate fork regression and participates in prereplicative repair of the damaged base in the reannealed duplex DNA [61, 62]. We compared the function of NEIL1 in stalling the replication fork at the damage sites for the prereplicative repair to the function of a "cow catcher" attached to the front of early steam locomotives that served to push aside animals or debris from the track ahead of the train's traversal, in a simplistic analogy to this exquisitely orchestrated process [63]. The key features of this "cow catcher" model are the ability of NEIL1 to recognize base lesions in ss DNA templates and its nonproductive binding to lesions in ss DNA, which, while preventing lethal DSB formation, causes the stalling of the replication fork. Subsequent fork reversal allows base lesion repair in the reannealed duplex. High expression and activation of NEIL1 in replicating cells, together with its stable physical and functional association with proteins in the DNA replication complex [48, 64–66], are consistent with this surveillance role of NEIL1. The human genome during each cell division may be at higher risk for oxidative damage whose repair would prevent accumulation of mutations in the daughter cells. Thus NEIL1's prereplicative BER function appears to be critical for preventing mutations and maintaining genome fidelity during cell division.

#### **6. Posttranslational modifications of BER proteins**

*In vitro* BER studies, carried out during the last couple of decades, are straightforward, mainly documenting functions of the repair proteins; however, in the complex cellular environment, the pathways are tightly regulated by interactions among the partner proteins in multiprotein complexes, which in turn also dictates the stability of the complexes. The stability and subcellular localization of these proteins are

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

regulated by site-specific posttranslational modifications (PTMs), primarily involving acetylation, methylation, phosphorylation, SUMOylation, ubiquitination, and PARylation. Thus PTMs are at the root of major regulatory processes, by bestowing novel biochemical properties to the modified proteins, including changes in enzymatic activity, subcellular localization, interaction partners, protein stability, and DNA binding. Although purified recombinant BER proteins without any PTMs are proficient in their enzymatic activities, *in cellulo* BER is significantly affected by these PTMs. In this section, we discuss all the major PTMs of BER proteins identified so far.

The hallmark of mammalian DGs and early BER proteins is the presence of nonconserved, intrinsically disordered appendages at the N or C terminus, which are absent in their bacterial orthologs. Some examples are the N-terminal extension in human NTH1 absent in the *E. coli* Nth, C-terminal extension in human NEIL1 which is lacking in *E. coli* Nei, N-terminal extension in human APE1 lacking in *E. coli* Xth [44, 65, 67, 68]. Although the unfolded sequence generally exists at the N or C terminus, this could also exist internally as in Human NEIL2, where it may serve as a linker of the two domains. Analogous to the situation of histones H3 and H4, where mostly all PTMs occur in the disordered N-terminal tail [69, 70], PTMs in many early BER proteins are clustered in their disordered domains. See **Table 2** for the major BER PTMs known so far.

### **6.1 Acetylation**

 Acetylation of histones was discovered back in 1963 after the Nobel prizewinning discovery of acetyl CoA [71–74], and acetylation of histones at the ε-amino group of Lys residues in their disordered N-terminal region was shown to suppress their abilities to inhibit transcription [75]. Following these pioneering discoveries that linked histone acetylation to chromatin decondensation and transcriptional activation [76–78], diverse acetylation modifiers were identified and characterized. These include various histone acetyltransferases (HATs) such as E1a-binding protein p300 (p300), CREB-binding protein (CBP), ortholog of yeast transcription regulator Gcn5, TAF(II)250 subunit of transcription factor IID, several members of the MYST family (MOZ, YBF2/SAS3, SAS2, and TIP60) and p300/CBP associated factor (PCAF). Histone deacetylases (HDACs) were subsequently discovered as "erasers," which include distinct members, HDACs1–11 and SIRTs in different transcriptional repressor complexes SIN3, NURD, etc., which regulate acetylation/ deacetylation cycle in cells [79–81]. These discoveries set the stage for epigenetic regulation of gene expression. Simultaneously, the concept of "reader" proteins [80, 82] that specifically recognize acetylated Lys residues through their bromodomains was introduced in addition to the "writers" (HATs) and "erasers" (HDACs). Although the first discovered nonhistone protein acetylation dated back in 1997 for the tumor suppressor TP53 [83], the overwhelming numbers of nonhistone protein acetylation, particularly in large macromolecular complexes involved in chromatin remodeling, DNA repair, cell cycle, etc., were appreciated much later, after 2006, from mass spectrometric-based proteomic approaches, and provided the global scenario of "cellular acetylome" [81, 84–86].

### **6.2 Phosphorylation**

Although enzymatic phosphorylation of proteins was discovered in 1954 [87], phosphorylated protein was known much earlier, based on identification of phosphate in vitellin [88], followed by detection of phosphoserine in this protein [89]. During the 1950s, ATP was discovered to be required for phosphorylation when the phosphate group was found to be covalently attached to specific serine/




**Table 2.**  *PTMs of BER proteins.*  threonine residues [90, 91]. Subsequently, various kinases that phosphorylate serine/threonine and later tyrosine residues were characterized for their ability to modulate protein functions [91–93]. As with acetylation, phosphorylation induces conformational changes in the protein that stimulates its enzymatic activity and modulates protein-protein interactions [92, 94, 95]. Although the initial studies in protein phosphorylation were focused on cellular communications and signal transduction pathways, eventually the critical role of protein kinases and the relevance of phosphorylation/dephosphorylation events in DNA damage response (DDR) are extensively acknowledged, and mass spectrometry-based global screening approaches enabled identification of diverse phosphorylation targets [96, 97].

#### **6.3 Ubiquitination and SUMOylation**

Proteins are also posttranslationally modified via isopeptide bond formation with small proteins, which leads to nonlinear polypeptides [98, 99]. Ubiquitin is the first-discovered and well-characterized member of this growing family of small peptide modifiers, which covalently modify diverse proteins involved in chromatin organization, gene expression, signal transduction, DDR, DNA repair, and protein degradation [100–102]. Ubiquitin signals are generated by an enzymatic cascade involving E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Ubiquitination is a highly dynamic process with deubiquitinases (DUBs) involved in this signaling, and growing evidence indicates the involvement of ubiquitination/deubiquitination in BER, as shown in **Table 2**.

Small ubiquitin-related modifier (SUMO), containing 100 amino acid (aa) residues protein, is ubiquitin-like polypeptide, which is conjugated to substrates in a manner similar to ubiquitination [102, 103]. The SUMO paralogs are synthesized as precursor proteins that are cleaved by a family of SUMO isopeptidases [104]. Mature SUMO is subsequently activated by a heterodimeric E1-activating enzyme Aos1/Uba2 (SAE1/SAE2) forming a thioester bond between its catalytic cysteine and the C-terminal carboxyl group of mature SUMO. Then SUMO is transferred to the catalytic cysteine of the E2-conjugating enzyme Ubc9. In contrast to the ubiquitin system where dozens of E2 enzymes have been identified, Ubc9 is the only known SUMO E2 conjugating enzyme. Finally, an isopeptide bond is formed between SUMO and the substrate by E3 ligases. A consensus SUMO acceptor site has been identified consisting of the sequence ΨKXE, where Ψ is a large hydrophobic amino acid and K is the site of SUMO conjugation [105]. There are at least four SUMO paralogs in humans, SUMO1, SUMO2, SUMO3, and SUMO4, which have more than 1000 protein targets. SUMOylation is highly dynamic and can be reversed by the action of deSUMOylating enzymes (SENPs). SUMOylation regulates protein-protein interactions involving SUMO-interacting motifs (SIMs), and it targets a group of proteins in the same pathway to facilitate association of multiprotein complexes for transcription, nuclear transport, chromatin assembly and modification, chromosome segregation, DNA damage repair, replication, and cell signaling [106, 107].

#### **6.4 PARylation**

 Poly ADP-ribosylation (PARylation), a crucial PTM that appears rapidly at DNA damage sites, is catalyzed by poly(ADP-ribose) polymerases (PARPs). The human PARP family contains 17 members among which only PARP1, 2, and 3 are involved in DDR [108–111]. PARPs covalently attach the ADP-ribose unit via an ester bond to the carboxyl group of glutamate or aspartate and sometimes also attach to cysteine or lysine of the target proteins [112–114]. PARPs successively

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

 transfer ADP-ribose units from NAD<sup>+</sup> to produce PAR chains containing up to 200 ADP-ribose units; however, in many cases, only single mono ADP-ribose moiety is transferred to the target proteins. Strand breaks in DNA activate PARP1, the founding and predominant member of the PARP family; the primary substrate of PARP1 is itself. Many proteins in the DDR pathways as well as the damage processing enzymes interact with PARP1 and/or are PARylated [112, 115]. In cells, PARylation/dePARylation is tightly and dynamically regulated; the PAR polymers are degraded by PAR glycohydrolase (PARG), possessing both exoglycosidic and endoglycosidic activities, and release free ADP-ribose moieties [116–118]. ADPribosyl-acceptor hydrolase (ARH) also exhibits PAR-degrading activity, although it has only exoglycosidase activity [119, 120]. Retention of PAR chains in cells triggers apoptotic cell death [121]. Although PARP1 interacts with the SSBR sensor XRCC1, as well as with other BER/SSBR proteins, and enables early recruitment of XRCC1 to the DNA lesions [122–126], there is no convincing evidence for PARylation of BER/SSBR proteins.

#### **6.5 Cross-talks between different PTMs, their regulation, and effect on BER**

Proteins employ diverse PTMs sequentially or concurrently to expand their repertoire of functions, thereby impacting global cellular signaling. The best example is the disordered N-terminal tail of histone H3, which has multiple sites for acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation [69, 70]. These PTMS could act synergistically or via reciprocal exclusion to modulate chromatin organization, thus affecting the transcriptome. The same Lys residues (K9, K27) in H3 are targets for both acetylation (marker of active chromatin) and methylation; however, monomethylation of these residues are markers of active chromatin, while di- and trimethylation are associated with repression. Recent evidence on BER enzymes, summarized below, suggests that specific modification at one site can dramatically influence another modification at a different site, which may critically impact BER activity.

Cyclin-dependent kinase (CDK)-mediated phosphorylation of UNG2 (nuclear UDG) in S phase signals its ubiquitination-dependent degradation, and CDKinhibitor roscovitine prevents such degradation [127, 128]. This suggests that phosphorylation-induced conformational change in UNG2 is a prerequisite for ubiquitination.

In the case of TDG, acetylation inhibits its repair activity by two distinct mechanisms. TDG acetylation at K94, K95, and K98 by p300/CBP suppresses BER by preventing APE1 recruitment to the damage site [129]. Protein kinase C (PKC) mediated phosphorylation at S93, S96, and S99, close to the acetylation sites, may promote repair by sterically blocking repair-inhibitory acetylation of adjacent lysine residues [130]. On the other hand, SUMOylation at K341 inhibits TDG's interaction with CBP, preventing its acetylation and thereby promoting BER [131].

 PARP1, SUMOylated at K203 and K486, is a target for ubiquitination and degradation, which is believed to be the mechanism for its turnover [132]. In contrast, PARP1's SUMOylation at K482 does not degrade the protein, rather stimulates PARylation of chromatin-associated proteins [133]. On the other hand, acetylation of PARP1, which stimulates its transactivation function, is inhibited by K486 SUMOylation. Thus, K486 SUMOylation restrains PARP1's transactivation function [134].

While acetylation of APE1 enhances its stability in chromatin and enzymatic activity [135, 136], CDK5-mediated phosphorylation enhances its ubiquitination and degradation [137, 138]. Thus, it is possible that phosphorylation and acetylation are mutually exclusive, acetylation stabilizing the protein, and phosphorylation guiding to its degradation.

In the case of PNKP, ATM-dependent phosphorylation was shown to prevent ubiquitination and hence its degradation. Thus, in response to oxidative stress, ATM phosphorylates and stabilizes PNKP in order to activate a coordinated DDR pathway [139–141]. Furthermore, PNKP interacts with the deubiquitination enzyme ataxin-3 (ATXN3), which enhances its stability and phosphatase activity [142].

Phosphorylation of FEN-1 by CDK1 at S187 was shown to promote SUMOylation at K168, which enhanced its polyubiquitination-dependent degradation [143]. Phosphorylation inhibits FEN-1's flap endonuclease activity [144, 145], which cross-talks with methylation, a lesser studied PTM of BER proteins. Methylation by arginine methyltransferase 5 at R192 prevents this phosphorylation and thus is proposed to be essential for the repair activities of FEN-1 [146]. Thus, in response to oxidative stress in cycling cells, methylation of FEN-1 could be a critical requirement for LP-BER.

 Acetylation of Pol β at K72 inhibits its dRP lyase activity [147], and this could account for acetylation-induced inhibition of enzymatic activity and switch from SN-BER to LP-BER. Methylation of Pol β at R137 has no effect on dRP lyase or DNA polymerase activities but inhibits its interaction with PCNA [148] and could thus be predicted to inhibit LP-BER. In contrast, R83 and R152 methylation enhanced Pol β's DNA binding and increased processivity [149]. Cellular Pol β level appears to be maintained by two ubiquitin E3 ligases, Mule and CHIP. DNA Pol β is monoubiquitinated by Mule, which in turn is recognized and polyubiquitinated by CHIP in undamaged cells. In response to oxidative stress, it is deubiquitinated, thus ensuring its stability and oxidized base damage repair [150, 151].

A recent study shows how PARylation stimulates SUMOylation [152]. In response to DNA strand breaks induced by alkylating agent methylmethanesulfonate (MMS), PARP1 is activated and synthesizes PAR chains; this promotes recruitment of SUMO E3 TOPORS to XRCC1, which facilitates XRCC1 SUMOylation. XRCC1 SUMOylation recruits Pol β at the damaged sites and thus ensures completion of BER.
