Figure 4.

Aag is the major Hx and ɛA DNA glycosylase in mouse liver, testes, kidney, and lung. (A) Release of ɛA and Hx by partially purified poly-histidine-Aag fusion protein (see Materials and Methods). 5′ ³²P-labeled double-stranded oligonucleotides containing site-specific ɛA or Hx were incubated with either polyhistidine-tagged Aag (lanes 2 and 4) or with negative control extracts from E. coli not expressing the Aag cDNA protein (see Materials and Methods) (lanes 1 and 3). After 1 hr incubation at 37°C to allow release of aberrant bases by Aag glycosylase, oligonucleotides were chemically cleaved at the abasic sites. Fragments were separated by 20% denaturing PAGE. Release of Hx or ɛA is indicated by the appearance of either a 14-mer or a 12-mer, respectively. (B) Liver: time-dependent release of Hx (Upper) and ɛA (Lower) for liver extract proteins from Aag +/+ mice (136 μg) and Aag −/− mice (142 μg). The BSA control or extract proteins were incubated at 37°C for the indicated times, chemically cleaved, and analyzed as described above. (C) Testes: 100 μg of extract proteins, prepared from the testes of two Aag −/− and two Aag +/+ mice were incubated for 5 hr with Hx and ɛA oligonucleotide substrates and analyzed as described above. (D) Kidney: 131 μg of extract proteins, prepared from the kidneys of two Aag −/− and two Aag +/+ mice, were incubated for 6 hr with Hx and ɛA containing oligonucleotide substrates and analyzed as described above. (E) Lung: 95 μg of extract proteins, prepared from the lungs of two Aag −/− and Aag +/+ mice, were incubated for 6 hr with Hx and ɛA oligonucleotide substrates and analyzed as described above.