Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES

John M Hinz,Peng Mao,Daniel R Mcneill,David M Wilson

doi:10.1074/jbc.m115.665547

Abstract

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

Highlights

The apurinic/apyrimidinic endonuclease 1 (APE1) endonuclease of base excision repair must access DNA damage within chromatin
WT APE1 activity has been previously reported to be reduced on nucleosomes relative to naked DNA, with the reduction being much greater when the AP site is inwardly oriented, with the backbone of the lesion facing toward the histone octamer [21]
Considering the high proportion of nuclear DNA associated with histones, we examined the possibility that the APE1 variants might exhibit different activities on substrate DNAs associated with nucleosome core particle (NCP)

Summary

Background

The apurinic/apyrimidinic endonuclease 1 (APE1) endonuclease of base excision repair must access DNA damage within chromatin. In comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced APDNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Our results in total suggest that APE1 harbors particular surface region residues that facilitate access to AP sites in the context of nucleoprotein complexes This finding may imply that BER proteins are equipped with a trait (termed “protein obstruction tolerance”) to cope with DNA damage embedded within protein-DNA complexes, alleviating the need for energy-driven processes (e.g. chromatin remodelers) for such high-frequency lesions

Experimental Procedures

Results

Discussion