**5. Intersection of posttranslational modifications and protein-protein interactions in BER coordination**

Posttranslational modifications (PTMs) of proteins involved in BER modulate catalytic and DNA-binding activities of individual proteins, their expression, intracellular localization, structure, and stability as well as protein-protein interactions and may therefore contribute to regulation of DNA repair either directly or indirectly. Numerous studies of PTMs and their functions in BER have been reviewed previously [90, 102–106]. As mentioned above, PARP1 modifies itself and binding partners with poly(ADP-ribose). Among the targets of PARP1 catalyzed ADP-ribosylation are two key BER proteins—XRCC1 and Polβ, and XRCC1 negatively regulates PARP1 activity [35, 107]. The automodification of PARP1 has been shown to enhance its interaction with XRCC1, LigIIIα, and TDP1; the length of

PAR polymer determines the efficiency of PAR-mediated accumulation of XRCC1 on DNA damage [46, 47, 108]. Recent studies have identified other PTMs, such as phosphorylation, acetylation, and methylation, to regulate the activity of PARP1 [104, 106]. Phosphorylation of PARP1 mediated by protein kinase CDK2 represents a novel DNA-independent mechanism of PARP1 activation [106]. Modifications of PARP1 mediated by AMP-activated protein kinase (AMPK) and lysine acetyltransferase 2B (PCAF) modulate both the activity of PARP1 and ADP-ribosylation of other proteins [106]. Polyubiquitination of PARP1 by E3 ubiquitin protein ligase is promoted by the automodification of PARP1 and targets PAR-PARP1 for proteasomal degradation [106]. It has to be noted that nonproteolytic roles of protein ubiquitination in regulation of DSBs repair and NER have been demonstrated [105]. PARP1 modification (at Lys486 residue) with small ubiquitin-like protein catalyzed by SUMO E3 ligase (SUMOylation) enhances p300-dependent acetylation of PARP1, while it has no effect on its activity [106].

The most abundant evidence on PTM-mediated regulation of protein-protein interactions is available for the XRCC1 protein. XRCC1 is an extensively phosphorylated protein with more than 45 phosphorylation sites localized in the linker regions and BRCTa domain [109]. Catalyzed by p38 MAPK kinase phosphorylation of the BRCTa domain (at T358 and T367 residues) has been shown to regulate PAR-mediated recruitment of XRCC1 to DNA damage [109]. The phosphorylation of XRCC1 by checkpoint kinase 2 (CHK2) at Thr284 residue *in vivo* and *in vitro*  increases the affinity of XRCC1 for DNA glycosylase MPG, facilitating thereby initiation of BER [110]. As shown recently, the same kinase interacts with PARP1 and modifies the BRCT domain; the CHK2-dependent phosphorylation of PARP1 stimulates its catalytic activity and interaction with XRCC1 [111]. Seven sites of XRCC1 phosphorylation mediated by kinase CK2 (localized in the XL2 linker) are necessary to modulate the interaction of XRCC1 with end-processing enzymes— PNKP, APTX, and PNK-like factor APLF—and the efficiency of repair of chromosomal DNA SSBs [37–39, 112, 113]. Notably, the phosphorylated and unmodified forms of XRCC1 bind different structural domains of PNKP and modulate the kinase activity of PNKP or its accumulation on DNA damage, respectively [37, 38]. The oxidized form of XRCC1 stabilized by a disulfide bridge between Cys12 and Cys20 residues forms a more stable (in comparison with the reduced form) complex with Polβ; an increase in the number of intermolecular contacts in this complex has been confirmed by X-ray analysis of the complex [23]. The existence of oxidized form of XRCC1 *in vivo* is essential to protect cells against extreme oxidative stress [114]. XRCC1 is a substrate for SUMOylation promoted by DNA damage-induced PARylation; SUMOylation of XRCC1 contributes to regulation of BER via increasing its binding affinity for Polβ [115].

The most frequent PTMs discovered for the multifunctional protein APE1 include phosphorylation, acetylation, S-nitrosylation, S-glutathionylation, formation of disulfide bonds, and ubiquitination [90, 102]. Most modifications modulate redox activity of APE1 and its regulatory function in transcription. As Cys residues are targets of different modifications, it is essential to understand the competition between these PTMs and their roles in APE1 function. Numerous studies on APE1 phosphorylation by a variety of protein kinases provide contradictory data on modulation of the repair activity of APE1 [102]. Recently, it has been shown that acetylation of APE1 (at Lys residues in the mammalian-conserved N-terminal extension) enhances both the AP-endonuclease activity and the interaction with XRCC1 and XRCC1-LigIIIα complex, ensuring cell survival in response to genotoxic stress [116].

Acetylation of DNA glycosylase TDG weakens its interaction with APE1 and produces opposite effects on the excision activity of the enzyme toward various

 types of base damages; repair of damage induced by the chemotherapeutic action of 5-fluorouracil is enhanced by the TDG acetylation [90]. Based on these data, the acetylation status of TDG within tumor cells was proposed to impact the chemotherapy efficacy. Phosphorylation of the flexible N-terminus of DNA glycosylase UNG2 (at Thr6 or Tyr8 residues) shown to disrupt interaction with the PCNA factor, without affecting the UNG2 catalytic activity or its RPA interaction, has been proposed to regulate the formation of the ternary PCNA-UNG2-RPA protein complex [117].

 Various PTMs of Polβ (acetylation, phosphorylation, and methylation) modulate its 5′-dRp-lyase and nucleotidyl transferase activities; the only example of PTMs impacts on protein-protein interaction is inhibition of Polβ-PCNA interaction due to PRMT1-dependent methylation of Arg137 [102]. The enzymes completing BER—LigIIIα and LigI—undergo posttranslational modification *in vitro* and in *vivo*; however, the intersection of their PTMs with protein-protein interactions is yet unknown [102].
