Abstract
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.
Highlights
A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity
Our results reveal that Ape1 requires some form of DNA secondary conformation for proficient ss AP site incision, that the type and location of the secondary structure with respect to the AP lesion can have a significant impact on Ape1 efficiency, and that Replication protein A (RPA) is likely a key negative regulator of Ape1 ss cleavage activity in vivo
Dependence of Ape1 ss Incision Activity on DNA Secondary Structure— Ape1 has been shown to effectively incise at AP sites in ssDNA [15], with its endonuclease efficiency seemingly influenced by the potential secondary structure of the oligonucleotide [16]
Summary
A major role of the multifunctional human Ape 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. Recent evidence argues that its AP endonuclease function is essential for cell viability [10, 11] For many years, this activity had been characterized on double-stranded (ds) APDNA substrates [12,13,14], as it was presumed that a successful BER event would take place exclusively on a template-containing (instructional) duplex DNA molecule. Our results reveal that Ape requires some form of DNA secondary conformation for proficient ss AP site incision, that the type and location of the secondary structure with respect to the AP lesion can have a significant impact on Ape efficiency, and that RPA is likely a key negative regulator of Ape ss cleavage activity in vivo
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