PMID:22819077

Citation	Yang, X, Fan, J, Ishchenko, AA, Patel, D, Saparbaev, MK and Ramotar, D (2012) Functional characterization of the Caenorhabditis elegans DNA repair enzyme APN-1. DNA Repair (Amst.) 11:811-22
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 apn1Δapn2Δtpp1Δ 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 apn1Δapn2Δtpp1Δ 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 apn1Δapn2Δtpp1Δ 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.
Links	PubMed Online version:10.1016/j.dnarep.2012.06.009
Keywords	Amino Acid Sequence; Animals; Caenorhabditis elegans/enzymology; Caenorhabditis elegans Proteins/chemistry; Caenorhabditis elegans Proteins/genetics; Caenorhabditis elegans Proteins/metabolism; DNA Breaks; DNA Repair; DNA Repair Enzymes/chemistry; DNA Repair Enzymes/genetics; DNA Repair Enzymes/metabolism; DNA, Fungal/genetics; DNA, Fungal/metabolism; DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism; Endodeoxyribonucleases/chemistry; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/metabolism; Glycine/genetics; Molecular Sequence Data; Nuclear Localization Signals/chemistry; Nucleotidases/genetics; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/genetics; Zinc/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CAEEL:APN1		GO:0006284: base-excision repair	ECO:0000247:	UniProtKB:P22936	P		Figure 1 shows C.elegans APN-1 shows homology to Yeast Apn1, which is involved in DNA repair	complete CACAO 8552
CAEEL:APN1		GO:0017005: 3'-tyrosyl-DNA phosphodiesterase activity	ECO:0000314:		F		Figure 10C shows that APN-1 exhibits a 3' repair diesterase activity	complete CACAO 8560
CAEEL:APN1		GO:0006284: base-excision repair	ECO:0000314:		P		Figure 10 shows that APN-1 has the ability to repair various DNA lesions	complete CACAO 8562
CAEEL:APN1		GO:0008311: double-stranded DNA 3'-5' exodeoxyribonuclease activity	ECO:0000314:		F		Fig.10C	complete CACAO 9044
CAEEL:APN1	enables	GO:0008311: double-stranded DNA 3'-5' exodeoxyribonuclease activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
CAEEL:APN1	involved_in	GO:0006284: base-excision repair	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
CAEEL:APN1	enables	GO:0017005: 3'-tyrosyl-DNA phosphodiesterase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
YEAST:APN1		GO:0006285: base-excision repair, AP site formation	ECO:0000314:		P		Fig.10. Incision activities of purified APN-1 on AP site and oxidized base lesions	complete CACAO 8801

References

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