Use of yeast for detection of endogenous abasic lesions, their source, and their repair.
Apurinic/apyrimidinic (AP) sites are expected to be one of the most frequent endogenous lesions in DNA. AP sites are potentially lethal and mutagenic. Data shows that the simultaneous inactivation of two AP endonucleases (Apn1 and Apn2) and of the nuclease Rad1-Rad10 causes cell death in Saccharomyces cerevisiae. We suggest that the essential function of Apn1, Apn2, and Rad1-Rad10 is to repair endogenous AP sites and related 3'-blocked single strand breaks. This data led us to conclude that the burden of endogenous AP sites is not compatible with life in absence of DNA repair. This chapter describes two genetic assays to investigate origin, repair, and biological consequences of endogenous AP sites in yeast. The first assay relies on genetic crosses and tetrad analysis and uses the apn1 apn2 rad1 triple mutant. The apn1 apn2 rad1 triple mutant is unviable; however, it can form microcolonies. By means of genetic crosses, apn1 apn2 rad1 x quadruple mutants are generated. The size of the colonies formed by each quadruple mutant is compared to that of the apn1 apn2 rad1 triple mutant. Three classes of genes (x) were identified: (i) genes whose inactivation aggravates the phenotype (reduces microcolony size), such as RAD9, RAD50, RAD51, RAD52, MUS81, and MRE11; (ii) genes whose inactivation alleviates the phenotype, such as UNG1, NTG1, and NTG2; and (iii) genes whose inactivation is neutral, such as MAG1 or OGG1. The second assay uses the apn1 apn2 rad14 triple mutant, which is viable but exhibits a spontaneous mutator phenotype. This mutant was used in a colethal screen. This assay allowed the identification of mutation in DNA repair genes such as RAD1 or RAD50, as well as a mutation in the DUT1 gene coding for the dUTPase, which has impact on the formation of AP sites in DNA. A model that summarizes our present and puzzling data on the origin and repair of endogenous AP sites is also presented.
Boiteux
Serge
S
Laboratory of Radiobiology DNA, Department of Radiobiology and Radiopathology, Aus Roses, France.
Guillet
Marie
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C083274:Apn1 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; C468953:APN2 protein, S cerevisiae; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D045643:DNA Repair Enzymes