**2. Base excision DNA repair**

The major pathway to remove damaged DNA bases is Base Excision Repair (BER, *Fig. 1*). BER can be divided into five steps: (i) excision of damaged base by the specific DNA glycosylase and formation of apurinic/apyrimidinic (AP) site; (ii) cleavage of phosphodiester bond at AP site by AP-endonuclease or AP-lyase; (iii) removal of chemical groups interfering with gap filling and ligation; (iv) gap filling; (v) ligation [2].

The first step of the BER pathway is recognition of damaged base by the specific DNA glycosylase, which cleaves N-glycosidic bond leaving behind a free base and an AP site. In humans about 10 DNA glycosylases of different, but partially overlapping substrate specificities are known [3]. Some of them are bifunctional enzymes, which have endowed AP-lyase activity and cleave phosphodiester bonds at 3' side of AP site either by ß- or ß/δ elimination. *E. coli* endonuclease III (Nth), its human homolog, hNTH1 and human 8-oxoG DNA glycosylase (OGG1) catalyse reaction of ß- elimination, which creates alpha/ßunsaturated aldehyde (3'dRP) at the 3' end of cleaved DNA strand. Bacterial formamidopyrimidine DNA glycosylase (Fpg), endonuclease VIII (Nei) and two human homologs of the latter, NEIL1 and NEIL2 catalyse ß/ δ-elimination and remove deoxyribose residue leaving phosphate at the 3' end of cleaved DNA strand. Monofunctional DNA glycosylases need the assistance of AP-endonucleases, which hydrolyse phosphodiester bond at the 5' end of the AP site. This yields DNA single strand break (SSB) with the 5'end

The Involvement of E2F1 in the Regulation of XRCC1-Dependent Base Excision DNA Repair 129

5'site of DNA break. In LP-BER a longer fragment ranging from 2 to 12 nucleotides is excised and re-synthesized [2]. Initially DNA polymerase elongates 3' end by a few nucleotides and moves aside a DNA fragment which contains 5' deoxyribophosphate. Subsequently, such flap structure is cleaved out by specific flap endonuclease, FEN1. It is believed that in LP-BER the first nucleotide is incorporated by DNA polymerase ß, while next ones by DNA polymerases δ or ε [2]. LP-BER demands also other assisting proteins,

The last stage of BER is ligation of repaired DNA fragments by DNA ligase. Different DNA ligases(LIG) are operating in short and long patch BER, LIG1 in LP-BER and LIG3alpha in SP-BER. LIG3alpha remains in complex with XRCC1 (x-ray repair cross-complementing

X-ray cross-complementing group 1 (XRCC1) is a 70- kDa protein comprising three functional domains; an N-terminal DNA binding domain, a centrally located BRCT I and a C-terminal BRCT II domain. It has no known enzymatic activity. Since it specifically interacts with nicked and gapped DNA *in vitro*[7-9], and rapidly and transiently responds to

DNA single-strand breaks (SSBs) are one of the most frequent types of DNA damage in cells [12]. SSBs can lead to the accumulation of mutations or can be converted from single to cytotoxic double-strand breaks. Thus, SSBs pose a critical threat to the genetic stability and survival of cells[13]. Various proteins have been identified that are part of the repair machinery for SSBs, including XRCC1 protein. XRCC1 has been shown to be critically involved in DNA SSB repair in studies using XRCC1-mutant cells and XRCC1 knockout mice[14], which have increased sensitivity to alkylating agents, ultraviolet and ionizing radiation [15], as well as elevated levels of sister chromatic exchange. Since XRCC1 interacts with many proteins known to be involved in BER and SSBR, it has been proposed that XRCC1 functions as a scaffold protein able to coordinate and facilitate the steps of various DNA repair pathways[11, 16]. For example, XRCC1 interacts with several DNA glycosylases involved in repair of both oxidative and alkylated base lesions, and stimulates their activity[17, 18]. This protein interacts with DNA ligase III, polymerase beta and poly (ADPribose) polymerase to participate in the base excision repair pathway. It is recruited to the site of DNA damage by several DNA glycosylases, e.g. OGG1 or NTH1 and remains at the site of repair till the last stage of ligation (*Fig. 2*), regulating and coordinating the whole process. XRCC1 facilitates exchange of DNA glycosylase with AP-endonuclease at the damaged substrate, which increases the excision rate of modified base, regulates pol ß interactions with APE1, and finally activates ligation step [17]. Binding of XRCC1 to Polynucleotide Kinase (PNK) enhances its capacity for damage discrimination, and binding of XRCC1 to DNA enables displacement of PNK from the phosphorylated product [19] thus accelerating SSBR of damaged DNA[20]. XRCC1 associates with Tyrosyl-DNA phosphodiesterase1(Tdp1) and enhances its activity required for repair of Top1-associated SSBs. It may act to recruit Tdp1 to these damaged sites[21]. Biochemical and nuclear magnetic resonance (NMR) experiments have demonstrated protein-protein interaction between the N-terminal domain of XRCC1 and the polymerase domain of pol β[22-25]. Additionally, stabilization of DNA ligase IIIα is dependent on its interaction with the BRCT II domain of XRCC1[26]. Aprataxin also interacts with XRCC1 and functions to maintain

PCNA (*proliferating cell nuclear antigen*) and RPC (*replication protein C*).

group 1) protein, which activates ligation of DNA ends by LIG3alpha.

**3. The role of XRCC1 protein in base excision DNA repair** 

DNA damage in cells, it may serve as a strand-break sensor [10, 11].

bearing baseless deoxyribose (5'dRP) and the 3' end with the free hydroxyl group. Both AP sites and SSBs can be formed due to spontaneous hydrolysis of purines, as well as upon DNA damaging agents, like ionizing radiation or oxidation.

Fig. 1. Base Excision Repair pathway

Before filling the gap by DNA polymerases possible additional chemical groups present on 3'OH end, which may block polymerisation, must be removed. Bacterial enzymes Xth (exonuclease III) and Nfo (Endonuclease IV), besides of cleaving phosphodiester bonds at 5' AP-site, have as well 3' phosphatase and 3' phosphodiesterase activities and remove phosphates and phosphoglycolates from 3' hydroxyl group of cleaved DNA strand[4]. In contrast, the major mammalian AP-endonuclease, APE1 effciently removes 3' phosphoglycolate groups, but has a very weak 3' phosphatase activity [5]. Phosphate groups left e.g. by NEIL1 glycosylase at 3'hydroxyls are most probably removed by polynucleotide kinase[6]. After cleavage of phosphodiester bond, repair may be continued on two alternative pathways (*Fig. 1*): *short-patch* BER (SP-BER) or *long patch* BER (LP-BER). During SP-BER in mammals, only one missing nucleotide is incorporated by DNA polymerase ß (pol ß), which has also endowed 5'dRPase activity and can remove baseless sugar from the

bearing baseless deoxyribose (5'dRP) and the 3' end with the free hydroxyl group. Both AP sites and SSBs can be formed due to spontaneous hydrolysis of purines, as well as upon

Before filling the gap by DNA polymerases possible additional chemical groups present on 3'OH end, which may block polymerisation, must be removed. Bacterial enzymes Xth (exonuclease III) and Nfo (Endonuclease IV), besides of cleaving phosphodiester bonds at 5' AP-site, have as well 3' phosphatase and 3' phosphodiesterase activities and remove phosphates and phosphoglycolates from 3' hydroxyl group of cleaved DNA strand[4]. In contrast, the major mammalian AP-endonuclease, APE1 effciently removes 3' phosphoglycolate groups, but has a very weak 3' phosphatase activity [5]. Phosphate groups left e.g. by NEIL1 glycosylase at 3'hydroxyls are most probably removed by polynucleotide kinase[6]. After cleavage of phosphodiester bond, repair may be continued on two alternative pathways (*Fig. 1*): *short-patch* BER (SP-BER) or *long patch* BER (LP-BER). During SP-BER in mammals, only one missing nucleotide is incorporated by DNA polymerase ß (pol ß), which has also endowed 5'dRPase activity and can remove baseless sugar from the

DNA damaging agents, like ionizing radiation or oxidation.

Fig. 1. Base Excision Repair pathway

5'site of DNA break. In LP-BER a longer fragment ranging from 2 to 12 nucleotides is excised and re-synthesized [2]. Initially DNA polymerase elongates 3' end by a few nucleotides and moves aside a DNA fragment which contains 5' deoxyribophosphate. Subsequently, such flap structure is cleaved out by specific flap endonuclease, FEN1. It is believed that in LP-BER the first nucleotide is incorporated by DNA polymerase ß, while next ones by DNA polymerases δ or ε [2]. LP-BER demands also other assisting proteins, PCNA (*proliferating cell nuclear antigen*) and RPC (*replication protein C*).

The last stage of BER is ligation of repaired DNA fragments by DNA ligase. Different DNA ligases(LIG) are operating in short and long patch BER, LIG1 in LP-BER and LIG3alpha in SP-BER. LIG3alpha remains in complex with XRCC1 (x-ray repair cross-complementing group 1) protein, which activates ligation of DNA ends by LIG3alpha.
