Molecular basis for processing of topoisomerase 1-triggered DNA damage by Apn2/APE2.

Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon.

Ribonucleotide Incorporation by Eukaryotic B-Family Replicases and Its Implications for Genome Stability.

Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.

How asymmetric DNA replication achieves symmetrical fidelity.

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3'-5' exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ's polymerization activity and MMR. Extrinsic proofreading across the genome is remarkably efficient. We report, with unprecedented accuracy, in vivo contributions of nucleotide selectivity, proofreading, and MMR to the fidelity of DNA replication in Saccharomyces cerevisiae. We show that extrinsic proofreading by Pol δ improves and balances the fidelity of the two DNA strands. Together, we depict a comprehensive picture of how nucleotide selectivity, proofreading, and MMR cooperate to achieve high and symmetrical fidelity on the two strands.

High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication.

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

Ribonucleotide incorporation into DNA during DNA replication and its consequences.

Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.

Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA.

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Metabolic and cardiovascular effects of chronic mild hyperuricemia in rodents.

Mildly elevated serum uric acid levels are common in people with metabolic syndrome and type 2 diabetes mellitus (T2DM), but whether elevated uric acid has a causal role in the pathogenesis of diabetes remains uncertain. We tested whether chronic mild hyperuricemia in rodents under controlled laboratory conditions can cause glucose intolerance in otherwise healthy animals, or whether it can worsen glucometabolic control in animals that are genetically predisposed to T2DM. We used an established model of experimental hyperuricemia in rodents with potassium oxonate dietary supplementation, which led to sustained, approximately two-fold elevation of uric acid compared with control animals. We also reversed the hyperuricemic effect of oxonate in some animals by treatment with a xanthine oxidase inhibitor. Manipulation of serum uric acid levels in Sprague-Dawley rats for up to 18 weeks did not affect fasting glucose and glucose tolerance. Blood pressure was also not affected by hyperuricemia in rats fed a Western-type diet. We next sought to determine whether uric acid may aggravate or accelerate the onset of glucometabolic abnormalities in rats already predisposed to T2DM. Chronic oxonate treatment in Zucker diabetic fatty (ZDF) and lean control rats for up to 6 weeks did not affect fasting glucose, insulin, and glucose tolerance in ZDF rats. Taken together, these findings indicate that elevated uric acid does not directly contribute to the pathogenesis of glucose intolerance and T2DM in rodents.

Studying Topoisomerase 1-Mediated Damage at Genomic Ribonucleotides.

Ribonucleotides incorporated into DNA by the DNA polymerases can be incised by Topoisomerase 1 (Top1) to initiate removal of ribonucleotides from the genome. This Top1-dependent ribonucleotide removal has been demonstrated to result in multiple forms of genome instability in yeast. Here, we describe both quantitative and qualitative assays to identify mutations and other forms of DNA damage resulting from Top1-cleavage at unrepaired genomic ribonucleotides.

Tumor physiological changes during hypofractionated stereotactic body radiation therapy assessed using multi-parametric magnetic resonance imaging.

Radiation therapy is a primary treatment for non-resectable lung cancer and hypoxia is thought to influence tumor response. Hypoxia is expected to be particularly relevant to the evolving new radiation treatment scheme of hypofractionated stereotactic body radiation therapy (SBRT). As such, we sought to develop non-invasive tools to assess tumor pathophysiology and response to irradiation. We applied blood oxygen level dependent (BOLD) and tissue oxygen level dependent (TOLD) MRI, together with dynamic contrast enhanced (DCE) MRI to explore the longitudinal effects of SBRT on tumor oxygenation and vascular perfusion using A549 human lung cancer xenografts in a subcutaneous rat model. Intra-tumor heterogeneity was seen on multi-parametric maps, especially in BOLD, T2* and DCE. At baseline, most tumors showed a positive BOLD signal response (%ΔSI) and increased T2* in response to oxygen breathing challenge, indicating increased vascular oxygenation. Control tumors showed similar response 24 hours and 1 week later. Twenty-four hours after a single dose of 12 Gy, the irradiated tumors showed a significantly decreased T2* (-2.9±4.2 ms) and further decrease was observed (-4.0±6.0 ms) after 1 week, suggesting impaired vascular oxygenation. DCE revealed tumor heterogeneity, but showed minimal changes following irradiation. Rats were cured of the primary tumors by 3x12 Gy, providing long term survival, though with ultimate metastatic recurrence.

The role of RNase H2 in processing ribonucleotides incorporated during DNA replication.

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 (rnh201-RED; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε (pol2-M644G) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 (rnh201Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2-5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication.

Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks.

Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2',3'-cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.

DNA Polymerases Divide the Labor of Genome Replication.

DNA polymerases synthesize DNA in only one direction, but large genomes require RNA priming and bidirectional replication from internal origins. We review here the physical, chemical, and evolutionary constraints underlying these requirements. We then consider the roles of the major eukaryotic replicases, DNA polymerases α, δ, and ɛ, in replicating the nuclear genome. Pol α has long been known to extend RNA primers at origins and on Okazaki fragments that give rise to the nascent lagging strand. Taken together, more recent results of mutation and ribonucleotide incorporation mapping, electron microscopy, and immunoprecipitation of nascent DNA now lead to a model wherein Pol ɛ and Pol δ, respectively, synthesize the majority of the nascent leading and lagging strands of undamaged DNA.

Processing ribonucleotides incorporated during eukaryotic DNA replication.

The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease.

Eukaryotic genome instability in light of asymmetric DNA replication.

The eukaryotic nuclear genome is replicated asymmetrically, with the leading strand replicated continuously and the lagging strand replicated as discontinuous Okazaki fragments that are subsequently joined. Both strands are replicated with high fidelity, but the processes used to achieve high fidelity are likely to differ. Here we review recent studies of similarities and differences in the fidelity with which the three major eukaryotic replicases, DNA polymerases α, δ, and ɛ, replicate the leading and lagging strands with high nucleotide selectivity and efficient proofreading. We then relate the asymmetric fidelity at the replication fork to the efficiency of DNA mismatch repair, ribonucleotide excision repair and topoisomerase 1 activity.

Stimulation of Chromosomal Rearrangements by Ribonucleotides.

We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase ε that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201Δ, rnh202Δ, and rnh203Δ), topoisomerase 1 (TOP1Δ), and/or carrying mutant alleles of DNA polymerases ε, α, and δ. We observed an ∼7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201Δ. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases α and δ, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication.

Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae.

Mismatches generated during eukaryotic nuclear DNA replication are removed by two evolutionarily conserved error correction mechanisms acting in series, proofreading and mismatch repair (MMR). Defects in both processes are associated with increased susceptibility to cancer. To better understand these processes, we have quantified base selectivity, proofreading and MMR during nuclear DNA replication in Saccharomyces cerevisiae. In the absence of proofreading and MMR, the primary leading and lagging strand replicases, polymerase ɛ and polymerase δ respectively, synthesize DNA in vivo with somewhat different error rates and specificity, and with apparent base selectivity that is more than 100 times higher than measured in vitro. Moreover, leading and lagging strand replication fidelity rely on a different balance between proofreading and MMR. On average, proofreading contributes more to replication fidelity than does MMR, but their relative contributions vary from nearly all proofreading of some mismatches to mostly MMR of other mismatches. Thus accurate replication of the two DNA strands results from a non-uniform and variable balance between error prevention, proofreading and MMR.

Measuring ribonucleotide incorporation into DNA in vitro and in vivo.

Ribonucleotides are incorporated into genomes by DNA polymerases, they can be removed, and if not removed, they can have deleterious and beneficial consequences. Here, we describe an assay to quantify stable ribonucleotide incorporation by DNA polymerases in vitro, and an assay to probe for ribonucleotides in each of the two DNA strands of the yeast nuclear genome.

Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.

Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.

Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair.

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.