Abstract
Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.
Highlights
Both physical and chemical factors in the cell and the environment can cause various types of DNA damage, which will cause some potential mutagenic and toxic effects on the cell
The potential AP endonuclease activity was tested using DNA carrying a synthetic AP site, dSpacer. On incubating both ssDNA and double-stranded DNA (dsDNA) with the purified TeuendoIV, a 17-nt DNA band, which is the product of the AP endonuclease, was generated (Figure 1b)
At the tested concentration of TeuendoIV, it generated a 16-nt DNA band, indicating that the 3 -exonuclease activity is possessed by TeuendoIV, which is similar to bacterial Endonuclease IV (EndoIV) [18,19]
Summary
Both physical and chemical factors in the cell and the environment can cause various types of DNA damage, which will cause some potential mutagenic and toxic effects on the cell. In addition to AP endonuclease activity, EndoIV and ExoIII have 3 -exonuclease activity, and function as 3 -repair diesterases by removing DNA 3 -blocking groups such as 3 -phosphates, 3 -phosphoglycolates and 3 -α,β unsaturated aldehydes [11,12]. On cleaving the DNA 5 to an AP site or removing the 3 -blocking groups, DNA polymerase will resynthesize a matched DNA strand by incorporating correct dNMPs into the 3 -OH end under the direction of a complementary template strand, and DNA ligase will seal the nick generated in the repair process [8] Though both EndoIV and ExoIII primarily function as an AP endonuclease and a 3 -repair diesterase, they have many contrasting properties. Our results provide biochemical information on repairing AP sites in hyperthermophilic archaea
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