Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is essential for the repair and resolution of approximately 20,000 base lesions per cell per day in both the nuclear and mitochondrial genomes [1], [2]. BER and single-strand break repair (SSBR) mechanisms facilitate repair of base damage and DNA single-strand breaks [3], [4]. This repair process helps avoid the accumulation of genome destabilizing base damage and BER intermediates which contribute to genomic instability, increased genetic mutations (base alterations, strand breaks), cancer development, and cancer progression [5], [6]. BER is a multiple step pathway defined by lesion recognition and incision (i.e., via a DNA glycosylase and APE1/PNKP), DNA gap tailoring (by DNA polymerase β, Polβ), and DNA synthesis and ligation (by Polβ and DNA ligases I and III) [7].

Polβ catalyzes two separate and essential enzymatic functions utilized during BER (5’dRP lyase for gap tailoring, and nucleotidyl transferase for DNA synthesis), and Polb knockout mice do not survive long after birth, highlighting its cellular importance [8], [9], [10], [11]. To facilitate lesion access and the rapid formation of BER protein complexes in chromatinized DNA, these reaction steps are coordinated following poly(ADP-ribose) (PAR) polymerase 1 (PARP1) and PARP2 activation and production of poly(ADP-ribose) (PAR) [12]. The subsequent formation of BER protein complexes are then assembled via the PAR-binding scaffold protein X-ray repair cross complementing 1 (XRCC1) [13], [14]. Through its PAR binding domain (PBD), XRCC1 binds to PAR and facilitates the recruitment of BER/SSBR factors including Polβ, APTX and DNA ligase III to sites of DNA damage. Importantly, the recruitment of Polβ is dependent on the interaction with XRCC1 via a defined protein-protein interaction domain [12], [15], [16].

We previously reported that disruption of the human Polβ/XRCC1 interaction led to diminished Polβ protein abundance [17]. Given the importance of Polβ on mouse survival, we herein investigated the role of the Polβ/XRCC1 interaction in a mouse model with targeted mutations in Polβ at the Polβ/XRCC1 interaction interface. We demonstrate that evolutionarily conserved Polβ amino acid residues L301 and V303 play a crucial role in mediating the Polβ/XRCC1 interaction in the mouse and in mouse cells. Disruption of L301 or V303 through a single mutation significantly reduces mouse Polβ protein recruitment to sites of DNA damage, while the dual mutation (Polβ L301R/V303R) does not bind XRCC1 and causes no detectable protein recruitment to sites of laser-induced DNA damage.

Numerous structures of human Polβ have been solved via X-ray crystallography [18], [19], [20], [21]. Here, for the first time, we studied the unliganded crystal structures of truncated wild-type (WT) mouse Polβ and the Polβ L301R/V303R dual mutation, each lacking the N-terminal 8 kDa lyase domain. Superposition of our mouse Polβ mutant model with the structure of rat Polβ protein complexed with the oxidized form of human XRCC1 [22] reveals that mutation of V303 to arginine would cause a direct clash with proline 2 of XRCC1, thereby leading to the likely destabilization of the Polβ/XRCC1 complex. Further, we generated CRISPR/Cas9-mediated Polβ L301R/V303R mice (PolbWT/L301R-V303R) and successfully bred them to homozygosity, suggesting loss of the Polβ/XRCC1 interaction is not necessary for survival, development, or fertility. Although the PolbL301R-V303R/L301R-V303R mice were smaller in size, they did not display any behavioral or stress-associated phenotypes, to-date. Finally, we verified that fibroblasts generated from PolbL301R-V303R/L301R-V303R mice do not show sensitivity to genotoxic stress but demonstrate significantly lower Polβ protein abundance similar to studies with human cells [17], [23].