In both budding and fission yeast, a large number of
ribonucleotides are incorporated into
DNA during replication by the major replicative polymerases (Pols α, δ and ɛ). They are subsequently removed by
RNase H2-dependent repair, which if defective leads to replication stress and
genome instability. To extend these studies to humans, where an
RNase H2 defect results in an
autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass
ribonucleotides during
DNA synthesis. In reactions containing
nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million
ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of
RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the
3'-exonuclease activity of yeast and human Pol δ largely fails to edit
ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses
ribonucleotides in
DNA templates, with four consecutive
ribonucleotides in
a DNA template being more problematic than single
ribonucleotides. In conjunction with recent studies in yeast and mice, this
ribonucleotide incorporation may be relevant to impaired development and disease when
RNase H2 is defective in mammals. As one tool to investigate
ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more
ribonucleotides into
DNA than does wild type Pol δ.