Quantitative characterization of protein–protein complexes involved in base excision DNA repair

Abstract

Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation

“…In this experimental setup, TDG will bind and immediately excise 5caC and then remain bound to the AP-site (Hardeland et al, 2002). We thus conclude that TDG interacts specifically with XRCC1-SUMO, which may also interact with, and carry along unmodified XRCC1 (Moor et al, 2015). Performing the experiment under exact same conditions with in vitro SUMOylated XRCC1 (50% XRCC1-SUMO), however, resulted in an efficient and specific binding of all, XRCC1-SUMO, POLb and DNA LIGIII to the TDG-bound substrate ( Fig 2D).…”

“…Next, to test if SUMO modification can mediate the formation of a BER complex on a DNA demethylation substrate, we assayed the concerted binding of TDG, XRCC1, POLb and LIGIII to biotinylated DNA duplexes containing a 5caC base. This, however, was observed only when unmodified XRCC1 was added in a mixture with SUMOylated XRCC1 (as produced upon in vitro SUMOylation) and can be explained by the tendency of XRCC1 to homodimerize (Moor et al, 2015), i.e., to form XRCC1-SUMO/ XRCC1 dimers in mixtures of modified and unmodified protein. Accordingly, TDG binding was obvious but neither XRCC1, nor POLb or LIGIII was detectable in the streptavidin precipitate of DNA substrate ( Fig 2C).…”

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair

“…These factors could play a key role in delivering XPC from its complex with PARP1 to the damage site in the absence of DDB2. Such events have been shown to play a role in dissociation of PARP1 from XRCC1 or APE1 during base excision repair (40,41).…”

Coordination of DNA single strand break repair

“…Interactions between specific BER/SSBR proteins and the PARP1 BRCT domain have recently been confirmed quantitatively by fluorescence-based approaches, which indicated similar binding affinities for PARP1 with XRCC1, POLβ and TDP1 [102, 103]. Of note, PARP1-XRCC1 and PARP1-POLβ binding affinities were not modulated by various BER intermediates, in contrast to the variable strength seen for the PARP1-APE1 interaction in the presence of different DNA substrates.…”