Nucleotide sequence and DNA secondary structure, as well as replication protein A, modulate the single-stranded abasic endonuclease activity of APE1.
A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity. This nuclease function has been well characterized on double-stranded (ds) DNA substrates, where the complementary strand provides a template for subsequent base excision repair events. Recently, Ape1 was found to incise efficiently at AP sites positioned within the single-stranded (ss) regions of various biologically relevant DNA configurations. The studies within indicated that the ss endonuclease activity of Ape1 is poorly active on ss AP site-containing polyadenine or polythymine oligonucleotides, suggesting a requirement for some form of DNA secondary structure for efficient cleavage. Computational, footprinting, and biochemical analyses indicated that the nature of the secondary structure and the proximity of the AP site influence Ape1 incision efficiency significantly. Replication protein A (RPA), the major ssDNA-binding protein in mammalian cells, was found to bind ss AP-DNA with similar affinity as unmodified ssDNA and ds AP-DNA with lower affinity. Consistent with their known relative DNA binding affinities, RPA blocks/inhibits the ss, but not ds, AP endonuclease function of Ape1. Moreover, RPA inactivates Ape1 incision activity at an AP site within the ss region of a fork duplex, but not a transcription-like bubble intermediate. The data herein suggested a model whereby RPA selectively suppresses the nontemplated ss cleavage activity of Ape1 in vivo, particularly at sites of ongoing replication/recombination, by coating the ssDNA.
Fan
Jinshui
J
Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, MD 21224, USA.
Matsumoto
Yoshihiro
Y
Wilson
David M
DM
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D051716:Replication Protein A / Q000502:physiology*; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D051716:Replication Protein A; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase