Apurinic/Apyrimidinic Endonuclease 1 and Tyrosyl-DNA Phosphodiesterase 1 Prevent Suicidal Covalent DNA-Protein Crosslink at Apurinic/Apyrimidinic Site

Abstract

Bifunctional 8-oxoguanine-DNA glycosylase (OGG1), a crucial DNA-repair enzyme, removes from DNA 8-oxo-7,8-dihydroguanine (8-oxoG) with following cleavage of the arising apurinic/apyrimidinic (AP) site. The major enzyme in eukaryotic cells that catalyzes the cleavage of AP sites is AP endonuclease 1 (APE1). Alternatively, AP sites can be cleaved by tyrosyl-DNA phosphodiesterase 1 (TDP1) to initiate APE1-independent repair, thus expanding the ability of the base excision repair (BER) process. Poly(ADP-ribose) polymerase 1 (PARP1) is a regulatory protein of DNA repair. PARP2 is also activated in response to DNA damage and can be regarded as the BER participant. Here we analyze PARP1 and PARP2 interactions with DNA intermediates of the initial stages of the BER process (8-oxoG and AP-site containing DNA) and their interplay with the proteins recognizing and processing these DNA structures focusing on OGG1. OGG1 as well as PARP1 and PARP2 form covalent complex with AP site-containing DNA without borohydride reduction. AP site incision by APE1 or TDP1 removal of protein adducts but not proteins' PARylation prevent DNA-protein crosslinks.

Keywords: 8-oxoguanine-DNA glycosylase; AP endonuclease 1; DNA-protein crosslinks; apurinic/apyrimidinic site; poly(ADP-ribose) polymerases; tyrosyl-DNA phosphodiesterase 1.

FIGURE 1

Influence of APE1, PARP2, and PARP1 on the crosslinking of OGG1 to AP site-containing DNA. Phosphorimages of crosslinked proteins to ³²P-labeled 8-oxoG-containing DNA (A) and to AP site-containing DNA after treatment with UDG (B). Reaction mixture included 10 nM DNA, 50 nM OGG1 and different concentrations of APE1 or PARP1/2 (50, 100, and 200 nM in A) and (10, 50, 100, and 200 nM in B). APE1, PARP1/2 were added to the reaction mixture simultaneously with OGG1.

FIGURE 2

Effect of the order of TDP1 and APE1 addition on the crosslinking of OGG1 to AP site-containing DNA. Phosphorimages of crosslinked proteins to ³²P-labeled 8-oxoG-containing DNA (A) and to AP site-containing DNA after treatment with UDG (B). Reaction mixture included 10 nM DNA, 50 nM OGG1 and different concentrations (50, 100, and 200 nM) of TDP1 or APE1. TDP1 or APE1 were added to the reaction mixture simultaneously with OGG1 (lanes 4–6 and 10–12) or after incubation of DNA with OGG1 for 10 min (lanes 7–9 and 13–15). Lane 1 in (A,B) represent DNA substrate incubated with 200 nM APE1, lane 2 – DNA substrate incubated with 50 nM OGG1, lane 3 – DNA substrate incubated with 200 nM TDP1.

FIGURE 3

Effect of NAD⁺ addition on the crosslinking of OGG1 to AP site-containing DNA in the presence of PARP1 (A) and PARP2 (B). Reaction mixture included 10 nM AP site-containing DNA after treatment with UDG, 50 nM OGG1 and different concentrations (50, 100, and 200 nM) of PARP1/2. NAD⁺ at final concentration 1 mM was added to the reaction mixture simultaneously (lanes 5–7) or after incubation of OGG1 with PARP1/2 for 10 min (lanes 8–10). The EMSA analysis of ternary OGG1-PARP2-DNA complex formation (C). AP-DNA after treatment with UDG was titrated by OGG1 (50, 100, and 200 nM) in the absence (lanes 2–4), in the presence of 100 nM PARP2 (lanes 8–10) or 100 nM PARP2 and 1 mM NAD⁺ (lanes 11–13). Lanes 5–7 – AP-DNA titration with PARP2 (50, 100, and 200 nM) in the absence of OGG1. Lane 1 is DNA control.

FIGURE 4

Poly(ADP-ribosyl)ation of OGG1 catalyzed by PARP1 or PARP2 in the presence of AP site-containing DNA. The reaction mixtures containing 100 nM PARP2 (A) or 100 nM PARP1 (B), 10 nM AP-DNA after treatment with UDG and 2.5 μM [³²P]-NAD⁺ were incubated at 25°C for 20 min in the presence 50 nM OGG1 with or without 100 nM APE1 and analyzed by SDS-PAG electrophoresis. Influence of APE1 on the OGG1 poly(ADP-ribosyl)ation by PARP2 in the presence of AP site-containing DNA (C). OGG1 at a final concentration 50 nM was added to the reaction mixture after PARP2 incubation with 10, 50, 100, and 200 nM APE1 (lanes 2–5) or simultaneously with APE1 (lanes 7–10).