FIGURE 3.

Unga functions to excise uracil bases from DNA. A and B, in vitro assay of uracil excision activity of Unga. The sequences of 20-bp double-stranded DNA oligonucleotides are shown in A. DNA substrates were incubated for 30 min at 37 °C with 30 ng/μl recombinant Unga or bacterial UDG, then stopped and separated on a polyacrylamide gel and stained (B). C and D, confocal fluorescence of incorporated BrdU. Embryos were co-injected at the one-cell stage with 1 pmol of BrdUTP and 500 pg of mcherry (mch) or 500 pg of unga-mcherry (unga-mch) mRNA, or 10 ng of cMO or 10 ng of unga-MO, and fixed at 64-cell and sphere stages for immunostaining with anti-BrdU and anti-ssDNA antibodies. E and F, relative intensities of BrdU signals. The BrdU incorporation level is reflected by the ratio of BrdU/ssDNA intensities in a nucleus. The ratios are shown in a string of crosses for all analyzed nuclei of the same treatment with an average indicated. Ne, number of observed embryos; Nc, total number of analyzed nuclei. ns, not statistically significant (p > 0.1).