Functional expression of Escherichia coli endonuclease IV in apurinic endonuclease-deficient yeast.
Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of abasic (AP) sites or oxidative DNA strand breaks. Yeast lacking Apn1 (apn1-) are hypersensitive to simple alkylating agents (which produce many AP sites) and to oxidants and display an elevated spontaneous mutation rate due to endogenous damages. We explored whether the prokaryotic repair enzyme could substitute for its yeast counterpart. Plasmid constructs were generated that expressed endonuclease IV at 1/20 to 10-fold the AP endonuclease activity of wild-type yeast; some of these plasmids expressed hybrid forms of endonuclease IV equipped with the C-terminal nuclear localization signal of Apn1. Although hybrid endonuclease IV-Apn1 (but not native endonuclease IV) was selectively localized to the yeast nucleus, expression of this chimeric protein at 25% of the normal Apn1 level did not restore alkylation or oxidant resistance to apn1- yeast, but it did partially counteract the mutator phenotype of apn1- yeast. Expression of either the hybrid protein or native endonuclease IV at approximately 10 times the wild-type Apn1 levels restored wild-type resistance to methyl methanesulfonate and near-wild-type H2O2 resistance. High level expression of native endonuclease IV also restored the normal spontaneous mutation rate to apn1- yeast. These data place limits on the amounts of AP endonuclease activity necessary for repair of DNA damages caused by both endogenous and environmental agents and point to a direct role of spontaneous AP sites as potentially mutagenic lesions.
Ramotar
D
D
CHUL, Health and Environment, Ste-Foy, Quebec, Canada.
Demple
B
B
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis* / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013489:Suppression, Genetic
D017931:DNA Primers; D029968:Escherichia coli Proteins; D008741:Methyl Methanesulfonate; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005947:Glucose; D005690:Galactose