Ribonucleotide incorporation in DNA by POLγ and in mtDNA from HeLa cells.
(A) Nucleotide sequence of the annealed DNA template. (B) Ribonucleotide incorporation by POLγ at increasing concentrations of all four rNTPs (0 μM, 40 μM, 100 μM, 400 μM, 1,000 μM of each) in the presence all four dNTPs (4 μM of each). Lanes 1–5, Alkali treated products; lanes 6–10, KCl treated samples (C) Cleavage products generated from alkaline and RNase H2 treatment (D) Alkaline treated or KCl treated products generated from extension assays with either POLγ or EXO-. (E) Cleavage products generated from extension assay with POLγ with the presence of indicated rNTP (4 mM) and all four dNTPs (4 μM of each). (F) Quantification of the ribonucleotide incorporation of POLγ, from the experiment in Fig 3D, as a percent of total product at every single position, inset: base composition of the incorporated ribonucleotide. (G) The same as in Fig 3F but the calculation is done with EXO- (H) Identity of incorporated ribonucleotide in H-strand (H) and L-strand (L) in HeLa cells. (I) Relative DNA base content in mtDNA of H-Strand (H) and L-strand (L). (J) Ribonucleotide incorporation frequency of an individual ribonucleotide per 1,000 complementary bases calculated on both H-strand and L-strand.