Identification of two apurinic/apyrimidinic endonucleases from Caenorhabditis elegans by cross-species complementation.
The Saccharomyces cerevisiae mutant strain YW778, which lacks apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase DNA repair activities, displays high levels of spontaneous mutations and hypersensitivities to several DNA damaging agents. We searched a cDNA library derived from the nematode Caenorhabditis elegans for gene products that would rescue the DNA repair defects of this yeast mutant. We isolated two genes, apn-1 and exo-3, encoding proteins that have not been previously characterized. Both APN-1 and EXO-3 share significant identity with the functionally established Escherichia coli AP endonucleases, endonuclease IV and exonuclease III, respectively. Strain YW778 expressing either apn-1 or exo-3 shows parental levels of spontaneous mutations, as well as resistance to DNA damaging agents that produce AP sites and DNA single strand breaks with blocked 3'-ends. Using an in vitro assay, we show that the apn-1 and exo-3 genes independently express AP endonuclease activity in the yeast mutant. We further characterize the EXO-3 protein and three of its mutated variants E68A, D190A, and H279A. The E68A variant retains both AP endonuclease and 3'-diesterase repair activities in vitro, yet severely lacks the ability to protect strain YW778 from spontaneous and drug-induced DNA lesions, suggesting that this variant E68A may possess a defect that interferes with the repair process in vivo. In contrast, D190A and H279A are completely devoid of DNA repair activities and fail to rescue the genetic instability of strain YW778. Our data strongly suggest that EXO-3 and APN-1 are enzymes possessing intrinsic AP endonuclease and 3'-diesterase activities.
Shatilla
Andrea
A
University of Montreal, Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre, 5415 de l'Assomption, Montreal, Que., Canada H1T 2M4.
Leduc
Anick
A
Yang
Xiaoming
X
Ramotar
Dindial
D
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D029742:Caenorhabditis elegans Proteins; D004269:DNA, Bacterial; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004706:Endodeoxyribonucleases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C504039:EXO-3 protein, C elegans; C102951:apn-1 protein, C elegans