**3. Proteins involved in BER interact directly with each other**

 Many protein participants of BER have been shown to interact physically with each other. Data on their direct interactions and structural domains involved are summarized in **Table 1**. Interactions of the XRCC1 protein with multiple partners have been explored in the greatest detail. The structure of XRCC1 is composed of three domains linked with disordered fragments (linkers XL1 and XL2), one of which (XL1) contains a nuclear localization signal (**Figure 3**) [23]. The availability of two BRCT domains (BRCTa and BRCTb) mediating protein-protein interactions (for review, see [24]), in addition to the N-terminal domain (NTD) involved in DNA binding, favors the main function of XRCC1 as scaffold in structural organization of "repairosomes". Interestingly, the binding sites of four enzymes catalyzing sequential steps of BER—APE1, PNKP (N-terminal domain), Polβ, and LigIIIα—are localized in different structural modules of XRCC1 (**Figure 3**). A second PNKP interaction site localized recently in XRCC1 (linker XL1) binds PNKP (catalytic domain) with lower affinity; this interaction has been proposed to stimulate PNKP activity, in contrast to the high-affinity interaction responsible for PNKP recruitment to DNA damage [25]. At the same time, the binding sites of various DNA glycosylases in XRCC1 overlap with those for APE1, Polβ, and PARP1 (**Figure 3**). It is likely that the enzymes initiating the repair of damaged bases form dynamic contacts with XRCC1 and other constituents of "repairosome." Direct interactions of DNA glycosylases NEIL1, NEIL2, and MYH with other enzymes of SP and LP BER (APE1, PNKP, Polβ, LigIIIα, Polδ, FEN1, and LigI) have been shown (**Table 1**). The multiprotein complexes of XRCC1 detected in many studies to be formed by recombinant proteins and cell extracts contain Polβ, PNKP, and LigIIIα as stable partners, and their presence enhances the interaction of XRCC1 with

#### **Figure 3.**

*The multidomain structure of XRCC1 and specific regions responsible for its scaffold function in BER. Protein partners and their binding sites in XRCC1 are shown schematically in the upper part of the figure. At the top, 3D structure models determined for the N-terminal domain, a fragment of XL2 linker, and the BRCTb domain crystallized as complexes with the respective domains of Polβ, PNKP, and LigIIIα (PDB codes: 3K75, 2W3O, and 3QVG) are presented. Reproduced with modification from [6] with permission of Pleiades Publishing, Ltd.* 

DNA glycosylases [30, 31, 33, 52]. PNKP and LigIIIα are the constituents of another multiprotein complex containing XRCC1 and TDP1 [53].

The PARP1 protein consists of multiple structural modules constituting an N-terminal DNA-binding domain and a C-terminal catalytic domain in addition to the central BRCT domain [55, 57]. The coordinating function of PARP1 in BER can be realized via direct interaction with some enzymes (PNKP, Polβ, LigIIIα, and TDP1) or indirect interaction mediated by the XRCC1 protein. The binding sites for main BER enzymes (Polβ and LigIIIα) and the scaffold XRCC1 protein are localized in the DNA binding and BRCT domains, while that for TDP1 is completed by the catalytic domain of PARP1 (**Table 1**). As a consequence, TDP1 is capable of the formation of a stable ternary complex with PARP1 and XRCC1 [53]. The overlapped binding sites for the majority of PARP1 partners create prerequisites for dynamic contacts in the preformed multiprotein assemblies, which can be stabilized in the complex with automodified PARP1 (PAR-PARP1). Many BER participants such as XRCC1, Polβ, PNKP, APTX, TDP1, LigIIIα, and LigI contain PAR-binding motifs, and some of them (XRCC1, LigIIIα, and TDP1) have been shown to interact with PAR-PARP1 more efficiently than with the unmodified PARP1 [46, 47, 58, 59]. The poly(ADP-ribose) acceptors have been identified in all the structural domains of PARP1; this expands significantly the platform for the formation of the "repairosomes" [60]. In contrast to PARP1, PARP2 does not have the BRCT domain and specialized zinc-fingers for DNA binding [15, 61]. The nonconserved WGR domain of PARP2 is responsible for the interaction with proteins (**Table 1**) as well as for DNA break detection [15]. The function of PARP2 (similar to that of PARP1) in coordination of the DNA repair process can be further mediated through its interaction with XRCC1 [17].


*a Protein domain(s) responsible for the interaction with protein partner(s) is shown in brackets. Structural composition of multidomain proteins: XRCC1: NTD 1–155, XL1 156–309, BRCTa 310–405, XL2 406–528, BRCTb 529–633; [23] PARP1: ZnF1 1–96, ZnF2 97–206, NLS 207–240, ZnF3 241–366, BRCT 381–484, WGR 518–661, CD 662–1014; [55] PARP2: NTD 1–63, WGR 64–198, CD 199–559; [36] LigIIIα: ZnF 1–100, linker 101–170, DBD 171–390, CD 391–836, BRCT 837–922 [56]. Designations: NTD/CTD, N-/C-terminal domain; CD, catalytic domain; DBD, DNA-binding domain; XL1/XL2, linker 1/2 in XRCC1 protein; NLS, nuclear localization signal; ZnF, zinc finger; FHA, forkhead-associated domain. The data for human and mouse (PARP2) recombinant proteins are presented.*

*b Techniques used in studies: affinity coprecipitation [25, 26, 29–36, 40, 41, 44–50], two-hybrid analysis [27, 30, 31, 35, 37, 46, 51–54], gel filtration [27, 28, 41, 42], ultracentrifugation [27, 50], coimmunoprecipitation [29, 31–33, 36–39, 41, 43, 46, 47, 52–54], fluorescence titration [38], fluorescence polarization [39], surface plasmon resonance [41], small-angle X-ray scattering [42], X-ray crystallography [23, 42, 45], and NMR [48].* 

#### **Table 1.**

*Interactions between main proteins involved in BER.* 

Direct interactions between the enzymes catalyzing different, usually sequential, steps of the BER process have been demonstrated in several studies (**Table 1**). Interestingly, the enzyme of the final step of SP BER—LigIIIα has direct binding partners among the enzymes involved in both the initial and middle steps of the process (NEIL1, NEIL2, PNKP, and TDP1), utilizing the BRCT domain for the interaction. Data reported recently indicate the ability of this enzyme to control the assembly of multiprotein complexes on single-strand DNA damages similar to PARP1, thus suggesting a scaffolding function of LigIIIα in the coordination of BER [62].

Most interactions between proteins involved in BER have been detected using the affinity coprecipitation, two-hybrid analysis, and immunoprecipitation techniques (**Table 1**). These techniques provide no information on physicochemical, structural, and conformational parameters of the complexes, leaving open many questions on the mechanisms of their functioning, such as the relative contribution of the proteins to the formation of macromolecular associates and their stoichiometry, the roles of dynamic interactions, conformational changes, and DNA intermediates in the formation of functional assemblies. Information on the structural

#### *Coordination of DNA Base Excision Repair by Protein-Protein Interactions DOI: http://dx.doi.org/10.5772/intechopen.82642*

 organization of these complexes is very limited. The 3D structures determined by X-ray crystallography are known for the isolated domains/fragments of the XRCC1 protein in complexes with the respective domains of its stable partners Polβ, LigIIIα, and PNKP (**Figure 3**). It is interesting to note that the specific contact region of the XRCC1 protein with LigIIIα (not involved in XRCC1 homodimerization)—a polypeptide consisting of hydrophobic amino acid residues at the N-terminus of the BRCTb domain—was revealed in the X-ray study [42]. The binding sites localized in proteins by the traditional nonequilibrium techniques participate obviously in the most stable interactions. The available structural data are not sufficient to decipher the molecular mechanisms of BER coordination.

 Using quantitative equilibrium techniques—fluorescence titration and fluorescence (Förster) resonance energy transfer (FRET)—we have characterized several homo- and hetero-oligomeric complexes of various BER proteins (**Figure 4**). *N*-hydroxysuccinimide esters of 5(6)-carboxyfluorescein (FAM) and 5(6)-carboxytetramethylrhodamine (TMR) were used for N-terminal fluorescent labeling of proteins. Direct (not mediated by DNA or other proteins) interactions of APE1 with Polβ, TDP1, and PARP1 and of Polβ with TDP1 as well as homooligomerization of APE1 have been detected for the first time. The apparent equilibrium dissociation constant (Kd) of the complexes is in the range of 23 to 270 nM. The XRCC1-PNKP complex characterized previously by using a similar approach has a Kd value in the same range [64]. The highest stability of the XRCC1 complex with Polβ was confirmed by the nonequilibrium approach, size exclusion chromatography coupled with multi-angle laser light-scattering (SEC-MALLS) [63]. Model DNAs imitating various DNA intermediates of BER have been shown to modulate the structure of protein complexes and their stability to different extents, depending on the type of DNA damage [63]. The DNA-dependent effects on the protein affinity for each other were most pronounced for the complexes of APE1 with different proteins (Polβ, XRCC1, and PARP1). Our findings advance understanding of the mechanisms underlying coordination and regulation of the BER process. The dependence of the efficiency of APE1 interaction with Polβ on the type of DNA intermediate indicates that functions of the two key enzymes are coordinated not only due to the differences in their affinity for DNAs as proposed previously in [65] but also due to the strength of their interaction with each other, which is controlled by DNA at different steps of repair. The higher affinity of APE1 for Polβ in the presence of AP-site containing DNA than in the complex with the incision product suggests that the efficient repair is facilitated by the transfer of the DNA intermediate to Polβ immediately during the incision step. The higher affinity of APE1 and Polβ for PARP1 than for each other in the presence of SSB containing DNA suggests that the regulation of functions of the BER participants via DNA-dependent modulation of their affinity for each other represents a common mechanism for various proteins. On the contrary, the stability of the XRCC1-Polβ complex does not depend on the presence of DNA intermediates, even though the most pronounced effect of different DNAs on the FRET signal, which reflects structural rearrangement of the complex, was detected for this complex. Our data indicate that this complex revealed in [66] to protect each protein from proteasome-mediated degradation may also serve as a stable component of the multiprotein assemblies, similar to the XRCC1-LigIIIα complex. Moreover, the XRCC1 binding sites with Polβ and LigIIIα do not overlap with regions mediating interactions with most other protein partners, thus enabling participation of the preformed ternary Polβ-XRCC1-LigIIIα complex in the entire Polβ- and XRCC1 dependent BER sub-pathway. Formation of the stable ternary complex *in vivo*  is evidenced by synchronous accumulation of XRCC1, Polβ, and LigIIIα at the damage sites of DNA [67, 68].

#### **Figure 4.**

*Direct interactions between BER proteins detected by fluorescence titration and FRET [63]. The EC50 values represent apparent equilibrium dissociation constants of the complexes (determined as half-maximal effective concentrations of protein partners); the length of black arrows connecting the protein pairs is proportional to the binding affinity; the underlined EC50 values have changed remarkably in the presence of DNA intermediates. The interaction in each pair of FAM- (donor) and TMR-labeled (acceptor) proteins is characterized by FRET efficiency (E); the highest change of the E value induced by DNA intermediates (increase/decrease with +/− sign) is presented in brackets. Reproduced with modification from [6] with permission of Pleiades Publishing, Ltd.* 

Recently, the oligomeric states of BER proteins and their complexes have been estimated based on hydrodynamic sizes determined by using dynamic light scattering (DLS) technique [69]. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner.

Interaction of PARP1 with Polβ and APE1 detected in our study [63] in both the absence and presence of DNA may contribute to regulation of the BER process. Cooperation between PARP1 and BER enzymes at different steps of DNA repair is evident from our previous studies. Interaction of PARP1, Polβ, and APE1 with the "central" DNA intermediate in BER established by photoaffinity labeling

*Coordination of DNA Base Excision Repair by Protein-Protein Interactions DOI: http://dx.doi.org/10.5772/intechopen.82642* 

 of BER proteins in the cell extract suggests interplay between these proteins during repair synthesis catalyzed by Polβ [70]. The ability of PARP1 to compete with APE1 for the binding of an AP-site containing DNA indicates possible cooperation between the proteins upon the recognition and further incision of the AP site [71]. Following the incision of AP site, PARP1 can catalyze the synthesis of poly(ADP-ribose). According to the initially proposed mechanism of its action, PARP1 dissociates from the complex with DNA after covalent attachment of the negatively charged PAR polymer. Further studies of an active role of PAR in the formation of the repair complexes have modified this hypothesis. It was established that following poly(ADP-ribosyl)ation, PARP1 was capable of covalent binding to the photoreactive DNA intermediate; the lifetime of such complexes was shown to depend on both the size of covalently bound PAR and the initial affinity of PARP1 for the DNA damage [70]. Complexes of PAR-PARP1 with damaged DNA have been detected by atomic force microscopy [72]. Recently, kinetics of poly(ADP-ribosyl)ation and PAR homeostasis (but not the PARP1 protein) have been proposed to play a primary role in protection of cells from acute DNA damage [73]. Hence, the formation of BER complexes on the damaged DNA can be regulated via either poly(ADP-ribosyl)ation of proteins or their interactions with PAR polymer synthesized by PARP1 and PARP2. Poly(ADP-ribose) is the most important cell regulator of protein-protein and protein-nucleic acid interactions. [20, 74–78].
