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.

DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.

Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.

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.

Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.

RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.

Ribonucleotides are signals for mismatch repair of leading-strand replication errors.

To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for repair of mismatches made by leading-strand DNA polymerase ε as compared to lagging-strand DNA polymerase δ. The results suggest that RNase-H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct mismatch repair to the continuously replicated nascent leading strand.

The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity.

The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

Opportunities for new studies of nuclear DNA replication enzymology in budding yeast.

Three major eukaryotic DNA polymerases, Polymerases α, δ, and ε (Pols α, δ, and ε), perform the fundamental process of DNA synthesis at the replication fork both accurately and efficiently. In trying to understand the necessity and flexibility of the polymerase usage, we recently reported that budding yeast cells lacking Pol ε exonuclease and polymerase domains (pol2-16) survive, but have severe growth defects, checkpoint activation, increased level of dNTP pools as well as significant increase in the mutation rates. Herein, we suggest new opportunities to distinguish the roles of Pol ε from those of two other eukaryotic B-family DNA polymerases, Pols δ and ζ.

Hypermutation signature reveals a slippage and realignment model of translesion synthesis by Rev3 polymerase in cisplatin-treated yeast.

Gene-gene or gene-drug interactions are typically quantified using fitness as a readout because the data are continuous and easily measured in high throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae and focusing on a matrix of DNA repair mutants and genotoxic drugs, we quantify 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not connected. Independent of fitness defects, we identified six cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted. One example occurred when yeast lacking RAD1 were exposed to cisplatin, and we characterized this interaction using whole-genome sequencing. Our sequencing results indicate mutagenesis by cisplatin in rad1Δ cells appeared to depend almost entirely on interstrand cross-links at GpCpN motifs. Interestingly, our data suggest that the following base on the template strand dictates the addition of the mutated base. This result differs from cisplatin mutation signatures in XPF-deficient Caenorhabditis elegans and supports a model in which translesion synthesis polymerases perform a slippage and realignment extension across from the damaged base. Accordingly, DNA polymerase ζ activity was essential for mutagenesis in cisplatin-treated rad1Δ cells. Together these data reveal the potential to gain new mechanistic insights from nonfitness measures of gene-drug interactions and extend the use of mutation accumulation and whole-genome sequencing analysis to define DNA repair mechanisms.

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.

Muver, a computational framework for accurately calling accumulated mutations.

BACKGROUND: Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. RESULTS: Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. CONCLUSIONS: Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.

Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools.

The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences.

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.

Differences in genome-wide repeat sequence instability conferred by proofreading and mismatch repair defects.

Mutation rates are used to calibrate molecular clocks and to link genetic variants with human disease. However, mutation rates are not uniform across each eukaryotic genome. Rates for insertion/deletion (indel) mutations have been found to vary widely when examined in vitro and at specific loci in vivo. Here, we report the genome-wide rates of formation and repair of indels made during replication of yeast nuclear DNA. Using over 6000 indels accumulated in four mismatch repair (MMR) defective strains, and statistical corrections for false negatives, we find that indel rates increase by 100 000-fold with increasing homonucleotide run length, representing the greatest effect on replication fidelity of any known genomic parameter. Nonetheless, long genomic homopolymer runs are overrepresented relative to random chance, implying positive selection. Proofreading defects in the replicative polymerases selectively increase indel rates in short repetitive tracts, likely reflecting the distance over which Pols δ and ϵ interact with duplex DNA upstream of the polymerase active site. In contrast, MMR defects hugely increase indel mutagenesis in long repetitive sequences. Because repetitive sequences are not uniformly distributed among genomic functional elements, the quantitatively different consequences on genome-wide repeat sequence instability conferred by defects in proofreading and MMR have important biological implications.

Mismatch repair balances leading and lagging strand DNA replication fidelity.

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to > 95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.

Genome-wide model for the normal eukaryotic DNA replication fork.

To investigate DNA replication enzymology across the nuclear genome of budding yeast, deep sequencing was used to establish the pattern of uncorrected replication errors generated by an asymmetric mutator variant of DNA polymerase δ (Pol δ). Sequencing of 16 genomes identified 1,206-bp substitutions generated over 33 generations by L612M Pol δ in a mismatch repair defective strain. Alignment of sequences flanking these substitutions identified "hotspot" motifs for Pol δ replication errors. The substitutions were distributed evenly across all 16 chromosomes. The vast majority were transitions that occurred with a strand bias that varied in a predictable manner relative to known functional origins of replication. This strand bias strongly supports the idea that Pol δ is primarily a lagging strand polymerase during replication across the entire nuclear genome.

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1.

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 A crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 A out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase zeta.

To probe Pol zeta functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol zeta in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol zeta is 30% as active as wild-type Pol zeta when replicating undamaged DNA. L979F Pol zeta shares with wild-type Pol zeta the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol zeta is error-prone compared to wild-type Pol zeta, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol zeta in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol zeta also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol zeta, and perhaps wild-type Pol zeta, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.