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Reference: Yang X, et al. (2012) Functional characterization of the Caenorhabditis elegans DNA repair enzyme APN-1. DNA Repair (Amst) 11(10):811-22

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Abstract

Caenorhabditis elegans possesses two distinct DNA repair enzymes EXO-3 and APN-1 that have been identified by cross-specie complementation analysis of the Saccharomyces cerevisiae apn1?apn2?tpp1? triple mutant deficient in the ability to repair apurinic/apyrimidinc (AP) sites and DNA strand breaks with blocked 3'-ends. While purified EXO-3 directly incises AP sites and removes 3'-blocking groups, such characterization has not been previously reported for APN-1. We recently documented that C. elegans knockdown for apn-1 is unable to maintain integrity of the genome. Despite the presence of EXO-3, the apn-1 knockdown animals are also defective in the division of the P1 blastomere, an observation consistent with the accumulation of unrepaired DNA lesions suggesting a unique role for APN-1 DNA repair functions. Herein, we show that C. elegans APN-1 is stably expressed as GST-fusion protein in S. cerevisiae only when it carries a nuclear localization signal, and with this requirement rescued the DNA repair defects of the S. cerevisiae apn1?apn2?tpp1? triple mutant. We purified the APN-1 from the yeast expression system and established that it displays AP endonuclease and 3'-diesterase activities. In addition, we showed that APN-1 also possesses a 3'- to 5'-exonuclease and the nucleotide incision repair activity. This latter activity is capable of directly incising DNA at the 5'-side of various oxidatively damaged bases, as previously observed for Escherichia coli endonuclease IV and S. cerevisiae Apn1, underscoring the importance of this family of enzymes in removing these types of lesions. Glycine substitution of the conserved amino acid residue Glu261 of APN-1, corresponding to Glu145 involved in coordinating Zn(2+) ions in the active site pocket of E. coli endonuclease IV, resulted in an inactive variant that lose the ability to rescue the DNA repair defects of S. cerevisiae apn1?apn2?tpp1? mutant. Interestingly, the Glu261Gly variant did not sustain purification and yielded a truncated polypeptide. These data suggest that the Glu261 residue of APN-1 may have a broader role in maintaining the structure of the protein.

Reference Type
Journal Article
Authors
Yang X, Fan J, Ishchenko AA, Patel D, Saparbaev MK, Ramotar D
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