Genomic DNA is susceptible to damage by endogenous and exogenous factors. Multiple DNA repair systems have evolved to protect against genetic mutation and instability [1]. Single-strand breaks (SSBs) and simple base lesions in DNA are repaired by SSB repair (SSBR) and the related base excision repair (BER) pathways, respectively [2], [3]. Apurinic/apyrimidinic (AP) or abasic sites are generated through spontaneous or enzymatic hydrolysis of the N-glycosyl bond between the deoxyribose and base. Enzymatic removal of a damaged base can occur through two glycosylase-initiated pathways [4]. In one pathway, a lesion-specific monofunctional DNA glycosylase removes the damaged base, and the resulting AP site is cleaved on its 5´-side by AP endonuclease 1 (APE1) creating a single-nucleotide gap. DNA polymerase β (pol β) conducts gap-filling DNA synthesis on the 3´-OH and removes the blocking 5´-deoxyribose group (dRP) in the gap generating a nicked substrate suitable for ligation [2]. In an alternate pathway, many of the glycosylases specific for oxidative DNA base damage are bifunctional with an associated AP lyase activity that cleaves the DNA backbone 3´ to the abasic site resulting in 3´-dRP and 5´-PO4 termini at the margins of the single-nucleotide gap. For example, the oxidatively damaged base, 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by 8-oxoG DNA glycosylase (OGG1). In this situation, APE1 can remove the 3´-blocking residue prior to DNA synthesis and ligation. Efficient repair of a BER-generated AP-site has been reconstituted in vitro with only three enzymes [5]. However, cellular DNA repair systems must rapidly respond to DNA damage within a diverse background of protein-associated DNA to avert the deleterious effects of altered DNA structure or cytotoxic DNA strand breaks on genomic stability [6]. Repair must exhibit versatility to target the correct enzymes needed for specific lesions at the appropriate time. This is accomplished through protein-protein interactions that are responsive to post-translational modifications, redox signaling events, as well as the cellular environment.

X-ray repair cross-complementing 1 (XRCC1) is a scaffold protein with no catalytic activity that interacts with a variety of SSBR and BER enzymes [7]. XRCC1 consists of three globular domains that are connected by two unstructured linkers. The XRCC1 amino-terminal domain binds pol β in a redox sensitive manner [8], while the carboxyl terminus domain includes a BRCA1 C-terminal (BRCT) domain (BRCT2) that binds DNA ligase IIIα. The central domain also includes a BRCT motif (BRCT1) that binds poly(ADP-ribose) (PAR) and mediates interactions with PARP1. Several other enzymes that tailor the termini in the gapped DNA prior to further repair interact with the linker between the BRCT motifs of XRCC1 in a phosphorylation-dependent manner [e.g., polynucleotide kinase/ phosphatase (PNKP) and aprataxin].

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein known to be an early responder in SSBR and BER [9]. It belongs to a family of at least 17 proteins; of these, only PARP1 and PARP2, and perhaps PARP3 are known to be active as DNA damage sensors [10], [11], [12]. PARP1 detects DNA strand breaks via its N-terminal zinc finger domains and is activated for PAR synthesis on itself and many other proteins involved in DNA repair and chromatin stability [13]. PARP1 auto-PARylation promotes recruitment of XRCC1 through binding to its BRCT1 domain. In addition, the negative charge of PAR polymers facilitates the dissociation of PARP1 from DNA and hastens repair. Thus, PARP catalytic inhibitors (PARPi) increase the PARP1/DNA lifetime leading to cytotoxic PARP1/DNA complexes that can result in replication-induced double-strand breaks (DSBs) [14]. The combination of DNA methylating agents with PARPi produce striking sensitization in cell culture studies [15], [16], [17], [18]. The discovery that PARP inhibition leads to synthetic lethality in cells with a deficiency in homologous recombination (e.g., mutations in breast cancer genes BRCA1 and BRCA2) has resulted in extensive development of potent PARP catalytic inhibitors as single agents for cancer treatment [19]; four PARPi are currently FDA-approved for clinical use (reviewed in [20], [21]).

Laser micro-irradiation-induced DNA damage is a method for rapidly creating localized DNA lesions that permits detection of repair protein recruitment in living cells [22]. Utilizing a low-energy irradiation protocol known to produce SSBs, transiently expressed and fluorescently-tagged pol β, XRCC1 and PARP1, amongst other repair proteins, have been characterized for recruitment [23], [24], [25], [26], [27]. To detect oxidized base damage and observe APE1 recruitment, an increase in the total energy of micro-irradiation was necessary [26]. Further increases in the energy of micro-irradiation generate DSBs. Previous reports have primarily described DNA repair protein recruitment in wild-type or complemented cell lines [25]. Additionally, other studies that have examined the recruitment of the BER factors considered here, focus on the level of recruitment and the subsequent loss of repair foci (i.e., dissociation of the fluorescently-tagged protein from localized DNA damage) [27]. Since quantitative recruitment kinetics are lacking in the literature, we aimed to compare the recruitment of three key transiently transfected fluorescently-tagged BER repair factors (pol β, XRCC1, and PARP1) in gene-deleted mouse embryonic fibroblasts with those expressing either endogenous or stably expressed repair factor. The analyses provide insights on the temporal order of binding of these key proteins as well as whether their endogenous levels, spectrum of DNA lesions generated by alternate micro-irradiation protocols, and PARP inhibition alters their recruitment kinetics. The results highlight the versatility of SSBR/BER for repair of heterogeneous lesions as revealed by both the similarity and differences in their recruitment and indicates that cellular DNA repair of ‘simple’ DNA lesions is multi-faceted.