Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase δ

Abstract

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.

Introduction

The presence of a ribonucleotide in DNA is potentially problematic because the 2′-oxygen on the ribose renders the DNA backbone susceptible to cleavage and potentially changes the conformation of the sugar pucker. Thus shortly after the discovery of DNA polymerases [1], it became of interest to examine how effectively DNA polymerases prevent ribonucleotide incorporation during DNA synthesis. Numerous studies since then (reviewed in [2], [3]) have revealed that discrimination against ribonucleotide incorporation can be high, but varies widely among DNA polymerases and is not absolute. The probability of ribonucleotide incorporation is increased by the fact that in both budding yeast [4] and in mammalian cells [5], ribonucleoside triphosphate (rNTP) concentrations are higher than deoxyribonucleoside triphosphates (dNTPs) concentrations. These facts led us to examine the frequency of stable ribonucleotide incorporation into DNA by the three DNA polymerases that replicate the budding yeast nuclear genome, Pols α, δ, and ɛ. In reactions containing physiological concentrations of the rNTPs and dNTPs, these replicases incorporate a surprisingly large number of ribonucleotides into DNA [4]. The biological relevance of these ribonucleotides was revealed by deleting the budding yeast RNH201 gene that encodes the catalytic subunit of RNase H2, the enzyme that initiates removal of ribonucleotides from DNA (see [6], [7] and references therein). Budding yeast rnh201Δ strains accumulate large numbers of ribonucleotides in their genome and they have several phenotypes characteristic of replicative stress, including genome instability [6], [7], [8], [9]. Large numbers of ribonucleotides are also incorporated by Pol ɛ into DNA in fission yeast, and these are also removed in a RNaseH2-dependent manner [10]. Moreover, Pol ɛ from budding yeast can proofread incorporated ribonucleotides, albeit not as efficiently as a misincorporated base [11].

The phenotypes of RNase H2-defective yeast may be relevant to the fact that defects in RNase H2 in humans result in Aicardi–Goutières syndrome, a recessive neuroinflammatory condition with similarities to autoimmune diseases [12]. It is therefore of interest to know whether the causes and consequences of ribonucleotide incorporation during DNA replication in yeast extend to higher eukaryotes. Of particular relevance here is a recent study demonstrating that more than a million ribonucleotides are present in the genome of RNase H2-defective mouse embryo cells [13]. These could result from failure to completely remove RNA primers from Okazaki fragments and/or from rNMPs incorporated by DNA polymerases during replication. As an initial step toward understanding the origins of ribonucleotides in the mammalian nuclear genome, and their possible relevance to human biology, we describe the ability to incorporate and proofread ribonucleotides during DNA synthesis in vitro by human Pol δ, which has been inferred to be the primary lagging strand replicase [14] and which has high fidelity and can efficiently proofread base-base mismatches [15]. We also examine Pol δ bypass of single and multiple consecutive ribonucleotides in DNA templates. We observe pausing during bypass that may contribute to the replicative stress observed in RNase H2-defective cells. Finally, having previously showed that a Leu612Met variant of yeast Pol δ that has lower than normal fidelity [14] also incorporates increased numbers of ribonucleotides into DNA, here we demonstrate that a similar variant in human Pol δ incorporates large amounts of ribonucleotides into DNA. Overall, the results suggest that the biochemical properties of yeast Pol δ regarding ribonucleotide processing are conserved in human Pol δ, which has several implications that are discussed below.