TABLE 2

DNA nuclease activity in S. cerevisiae

StrainSubstrateTCA-soluble nucleotides (pmol)
MF21/pPM29ssDNA1
MF21/pPK3(DIN7)ssDNA31
MF21/pPM29dsDNA7
MF21/pPK3(DIN7)dsDNA24
  • Strain MF21 exo1::URA3 harboring pPK3, which carries DIN7 sequence fused to the hexahistidine-tag sequence and the expression of which is under control of the GAL1 promoter, and MF21 carrying vector pPM29 were grown in YNBG medium. Then, galactose was added for an additional 6 hr. Extracts were prepared and were partially purified with the use of metal ion chromatography as described in materials and methods. Reaction mixtures contained 5 μl (4.1 μg protein) of Ni-NTA agarose fractions eluted with 50 mm imidazole. Activity is expressed as picomoles of TCA-soluble nucleotides released.