Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institute of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
DNA Repair (Amst). 2013 Feb 1;12(2):121-7. doi: 10.1016/j.dnarep.2012.11.006. Epub 2012 Dec 12.
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.
在芽殖酵母和裂殖酵母中，大量的核糖核苷酸在复制过程中被主要的复制聚合酶（Pol α、δ 和 ɛ）掺入 DNA 中。随后，它们被 RNase H2 依赖性修复去除，如果修复有缺陷，会导致复制应激和基因组不稳定。为了将这些研究扩展到人类，在人类中，RNase H2 的缺陷会导致自身免疫性疾病，我们在这里比较了人类和酵母 Pol δ 在 DNA 合成过程中掺入、校对和绕过核糖核苷酸的能力。在含有核苷酸浓度估计存在于哺乳动物细胞中的反应中，人类 Pol δ 稳定地掺入一个 rNTP 约 2000 个 dNTP，这一比例与酵母 Pol δ 相似。这一结果表明，人类 Pol δ 每复制一轮可能会将超过 100 万个核糖核苷酸引入核基因组，这一数量最近在 RNase H2 缺陷型小鼠细胞的基因组中被报道存在。与如此大量的稳定掺入一致，我们表明，酵母和人类 Pol δ 的 3'-外切核酸酶活性在聚合过程中很大程度上不能编辑核糖核苷酸。我们还表明，与酵母 Pol δ 一样，人类 Pol δ 在绕过 DNA 模板中的核糖核苷酸时会暂停，在 DNA 模板中有四个连续的核糖核苷酸比单个核糖核苷酸更成问题。结合最近在酵母和小鼠中的研究，当 RNase H2 在哺乳动物中缺陷时，这种核糖核苷酸的掺入可能与发育受损和疾病有关。作为一种研究人类细胞中 Pol δ 掺入核糖核苷酸的工具，我们表明，在聚合酶活性位点含有 Leu606Met 取代的人类 Pol δ 将 7 倍以上的核糖核苷酸掺入 DNA 中，比野生型 Pol δ 多。
