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Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase delta

Journal article
Authors Anders R Clausen
S. Zhang
P. M. Burgers
M. Y. Lee
T. A. Kunkel
Published in DNA Repair (Amst)
Volume 12
Issue 2
Pages 121-7
ISSN 1568-7856
Publication year 2013
Published at
Pages 121-7
Language en
Links dx.doi.org/10.1016/j.dnarep.2012.11...
Keywords DNA Polymerase III/genetics/*metabolism, *DNA Repair, DNA Replication, Humans, Mutation, Ribonucleotides/*metabolism, Saccharomyces cerevisiae/enzymology
Subject categories Biochemistry and Molecular Biology

Abstract

In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols alpha, delta and varepsilon). 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 delta to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol delta stably incorporates one rNTP for approximately 2000 dNTPs, a ratio similar to that for yeast Pol delta. This result predicts that human Pol delta 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 delta largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol delta, human Pol delta 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 delta in human cells, we show that human Pol delta containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol delta.

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