Abstract
Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in Ciona intestinalis. Amino acid sequence analysis suggested that CiAPEX2 and CiP0 are AP endonuclease homologues. Although we could not detect AP endonuclease or 3′-phosphodiesterase activity, these two purified proteins exhibited 3′-5′ exonuclease activity. This 3′-5′ exonuclease activity was sensitive to ethylenediaminetetraacetic acid (EDTA), and the efficiency of this activity was influenced by the 3′-terminus of substrate DNA. Both CiAPEX2 and CiP0 degraded not only a 5′-protruding DNA end, but also nicked DNA, which is generated through AP endonuclease 1 (APEX1) cleavage. These two genes partially complemented the growth rate of AP endonuclease-deficient Escherichia coli treated with hydrogen peroxide. These results indicate that 3′-5′ exonuclease activity is an evolutionarily conserved enzymatic activity of APEX2 and P0 homologues and this enzymatic activity may be important for AP endonucleases.
Highlights
Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions, with approximately 9000 AP sites per cell produced per day in mammalian cells [1]
To clarify the differences of the roles and functions among AP endonucleases, we focused on the homologues of AP endonuclease 2 (APEX2) (CiAPEX2) and P0 (CiP0) in C. intestinalis
This matched DNA preference is the same for tagfree and His-tagged proteins (Additional file 2: Figure S2). These results indicate that CiAPEX2 and CiP0 are not likely to contribute to the editing function, in contrast to previous studies which suggested that 3′-5′ exonuclease activity may work as an editing function [14]
Summary
Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions, with approximately 9000 AP sites per cell produced per day in mammalian cells [1] These DNA damages are generated via spontaneous depurination and base removal by DNA glycosylases [1,2,3]. Among P0 homologues, only Drosophila melanogaster (D. melanogaster) P0 homologue (DmP0) is reported to have AP endonuclease and DNase activity [21]. H. sapiens P0 homologue is reported to interact with HsAPEX1 [23] Considering these facts, P0 homologues are ribosomal proteins, they are expected to adopt 3Dstructures like that of AP endonuclease and participate in AP site repair. To clarify the differences of roles among AP endonucleases, we focused on two candidate AP endonucleases in C. intestinalis, CiAPEX2 and CiP0. We report the results of amino acid sequence analysis, in vitro experiments using purified CiAPEX2 and CiP0, and complementation assays
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