Impact of abasic site orientation within nucleosomes on human APE1 endonuclease activity

Abstract

Glycosylases responsible for recognizing DNA lesions and initiating Base Excision Repair (BER) are impeded by the presence of histones, which are essential for compaction of the genetic material in the nucleus. Abasic sites are an abundant mutagenic lesion in the DNA, arising spontaneously and as the product of glycosylase activity, making it a common intermediate in BER. The apurinic/apyrimidinic endonuclease 1 (APE1) recognizes abasic sites and cleaves the DNA backbone adjacent to the lesion, creating the single-strand break essential for the subsequent steps of BER. In this study the endonuclease activity of human APE1 was measured on reconstituted nucleosome core particles (NCPs) with DNA containing enzymatically-created abasic sites (AP) or the abasic site analog tetrahydrofuran (TF) at different rotational positions relative to the histone core surface. The presence of histones on the DNA reduced APE1 activity overall, and the magnitude was greatly influenced by differences in orientation of the lesions along the DNA gyre relative to the histone core. Abasic moieties oriented with their phosphate backbones adjacent to the underlying histones (In) were cleaved less efficiently than those oriented away from the histone core (Out) or between the In and Out orientations (Mid). The impact on APE1 at each orientation was very similar between the AP and TF lesions, highlighting the dependability of the TF abasic analog in APE1 activity measurements in nucleosomes. Measurement of APE1 binding to the NCP substrates reveals a substantial reduction in its interaction with nucleosomes compared to naked DNA, also in a lesion orientation-dependent manner, reinforcing the concept that reduction in APE1 activity on nucleosomes is due to occlusion from its abasic DNA substrate by the histones. These results suggest that APE1 activity in nucleosomes, like BER glycosylases, is primarily regulated by its chance interactions with transiently exposed lesions.

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes

“…Notably, the reverse primer contains a 5Ј biotin tag, preventing labeling of the strand without the uracil residues during radiolabeling. Uracil residues were subsequently converted to abasic (AP) sites as described previously (21), by treatment of the uracil DNAs with recombinant Escherichia coli UDG (New England Biolabs) for 30 min (reaction buffer: 25 mM HEPES (pH 7.5), 2 mM DTT, 0.2 mM EDTA, 100 g/ml of BSA, 10% glycerol, 5 mM MgCl 2 , 4 mM ATP). Mononucleosomes (NCPs) were prepared by histone octamer transfer as described previously (18,21).…”

“…Uracil residues were subsequently converted to abasic (AP) sites as described previously (21), by treatment of the uracil DNAs with recombinant Escherichia coli UDG (New England Biolabs) for 30 min (reaction buffer: 25 mM HEPES (pH 7.5), 2 mM DTT, 0.2 mM EDTA, 100 g/ml of BSA, 10% glycerol, 5 mM MgCl 2 , 4 mM ATP). Mononucleosomes (NCPs) were prepared by histone octamer transfer as described previously (18,21). Three pmol of radiolabeled 147-bp DNA substrates were combined with 300 pmol of chicken erythrocyte core particles prepared from chicken erythrocytes (29) at high ionic strength, and reconstituted by subsequent incremental dialysis (30) (Fig.…”

“…The reaction buffer, consisting of 25 mM HEPES (pH 7.5), 2 mM DTT, 0.2 mM EDTA, 100 g/ml of BSA, 10% glycerol, 5 mM MgCl 2 , and 4 mM ATP was identical to that used previously to assess APE1 activity on NCPs (21). To adjust for excess core particles present in reconstituted nucleosome samples, naked DNA samples had chicken erythrocyte core particles added to a final concentration of 300 pM.…”

“…APE1 incision activity is greatly reduced (ϳ10-fold) on nucleosomes at an AP site with its backbone sugar moiety in an "inward" orientation relative to an "outward" oriented AP site (21). Notably, APE1 activity on the outward-oriented AP site in nucleosomes was much lower than that on naked DNA (ϳ10-fold), highlighting the impact of the histone proteins on APE1, even when they are not directly occluding the lesion.…”

“…The enzymatic activities of APE1 have been well characterized on a wide range of abasic DNA substrates, with a few studies examining its functionality in the context of nucleosomes (21,22). APE1 incision activity is greatly reduced (ϳ10-fold) on nucleosomes at an AP site with its backbone sugar moiety in an "inward" orientation relative to an "outward" oriented AP site (21).…”