Figure 2.
Stable incorporation of ribonucleotides into DNA by proofreading-deficient human (h) and yeast (y) Pol δ. (A) Alkali cleavage products of reactions with all eight NTPs at cellular concentrations by human and yeast Pol δ in the presence of PCNA. The relative amount of ribonucleotides incorporated into the primer strand is indicated below each lane. L indicates the ladder. (B) Average frequency of ribonucleotide incorporation by human Pol δ according to the incorporated ribonucleotide. (C) Average frequency of ribonucleotide incorporation by yeast Pol δ according to the incorporated ribonucleotide. (D) Frequency of ribonucleotide incorporation by human Pol δ at each of 24 template positions. (E) Frequency of ribonucleotide incorporation by yeast Pol δ at each of 24 template positions. (F) Primed RPA-coated SS-M13mp18 DNA was fully replicated by proofreading proficient and deficient yeast Pol δ or the Leu612Met mutant forms under standard conditions with or without rNTPs, as described before (6). Reactions were treated with 0.3 M NaOH. The products were recovered by ethanol precipitation and separated on a 1% alkaline agarose gel. Median product sizes are indicated below the gel image. Results are from at least two independent experiments.