Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

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.

A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity.

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.