Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3′–5′ exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.     

Characterization of Family D DNA polymerase from Thermococcus sp. 9°N

Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we report the biochemical characterization of a heterodimeric polD from Thermococcus. PolD contains both DNA polymerase and proofreading 3′–5′ exonuclease activities to ensure efficient and accurate genome duplication. The polD incorporation fidelity was determined for the first time. Despite containing 3′–5′ exonuclease proofreading activity, polD has a relatively high error rate (95 × 10^?5) compared to polB (19 × 10^?5) and at least 10-fold higher than the polB DNA polymerases from yeast (polε and polδ) or Escherichia coli DNA polIII holoenzyme. The implications of polD fidelity and biochemical properties in leading and lagging strand synthesis are discussed.     

Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1

Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.     

NAR Breakthrough Article: Telomere-associated proteins add deoxynucleotides to terminal proteins during replication of the telomeres of linear chromosomes and plasmids in Streptomyces

Typical telomeres of linear chromosomes and plasmids of soil bacteria Streptomyces consist of tightly packed palindromic sequences with a terminal protein (‘TP’) covalently attached to the 5′ end of the DNA. Replication of these linear replicons is initiated internally and proceeds bidirectionally toward the telomeres, which leaves single-strand overhangs at the 3′ ends. These overhangs are filled by DNA synthesis using the TPs as the primers (‘end patching’). The gene encoding for typical TP, tpg, forms an operon with tap, encoding an essential telomere-associated protein, which binds TP and the secondary structures formed by the 3′ overhangs. Previously one of the two translesion synthesis DNA polymerases, DinB1 or DinB2, was proposed to catalyze the protein-primed synthesis. However, using an in vitro end-patching system, we discovered that Tpg and Tap alone could carry out the protein-primed synthesis to a length of 13 nt. Similarly, an ‘atypical’ terminal protein, Tpc, and its cognate telomere-associated protein, Tac, of SCP1 plasmid, were sufficient to achieve protein-primed synthesis in the absence of additional polymerase. These results indicate that these two telomere-associated proteins possess polymerase activities alone or in complex with the cognate TPs.     

Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.     

Highly accurate synthesis of the fully 2′-fluoro-modified oligonucleotide by Therminator DNA polymerases

Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2′-fluoro-modified (2′F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2′F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.     

In vitro synthesis of Escherichia coli ribosomal RNA

Synthesis of ribosomal RNA in a cell-free system was achieved using purified Escherichia coli RNA polymerase and bacterial DNA templates from E. coli, Proteus mirabilis and E. coli/P. mirabilis hybrid strains carrying an E. coli DNA enriched for ribosomal RNA genes.Both direct and indirect competition hybridization revealed that from 5 to 15% of the in vitro product, depending on the template used, had sequences homologous to rRNA. The level of synthesis of sequences homologous to rRNA was related directly to the proportion of rRNA genes in the template. The use of heterologous DNA during competition hybridization ensured at least a 100-fold greater sensitivity for the detection of rRNA sequences than from any messenger RNA sequence.     

Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.     

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.     

De novo DNA synthesis using single molecule PCR

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.     

Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity

Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase κ (Polκ) encoded by the DINB1 gene. Our recent studies have indicated that human Polκ is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polκ also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polκ during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polκ synthesizes DNA with extraordinarily low fidelity. At the lacZα target gene, human Polκ made on average one error for every 200 nucleotides synthesized, with a predominant T→G transversion mutation at a rate of 1/147. The overall error rate of human Polκ is 1.7-fold lower than human Polη, but 33-fold higher than human Polβ, a DNA polymerase with very low fidelity. Thus, human Polκ is one of the most inaccurate DNA polymerases known. These results support a role for human Polκ in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.     

In vitro selections with RNAs of variable length converge on a robust catalytic core

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.     

Small circular DNAs for synthesis of the human telomere repeat: varied sizes, structures and telomere-encoding activities

We describe the construction, structural properties and enzymatic substrate abilities of a series of circular DNA oligonucleotides that are entirely composed of the C-rich human telomere repeat, (CCCTAA)_n. The nanometer-sized circles range in length from 36 to 60 nt, and act as templates for synthesis of human telomere repeats in vitro. The circles were constructed successfully by the application of a recently developed adenine-protection strategy, which allows for cyclization/ligation with T4 DNA ligase. Thermal denaturation studies showed that at pH 5.0, all five circles form folded structures with similar stability, while at pH 7.0 no melting transitions were seen. Circular dichroism spectra at the two pH conditions showed evidence for i-motif structures at the lower pH value. The series was tested as rolling circle templates for a number of DNA polymerases at pH = 7.3–8.5, using 18mer telomeric primers. Results showed that surprisingly small circles were active, although the optimum size varied from enzyme to enzyme. Telomeric repeats 1000 nt in length could be synthesized in 1 h by the Klenow (exo-) DNA polymerase. The results establish a convenient way to make long human telomeric repeats for in vitro study of their folding and interactions, and establish optimum molecules for carrying this out.     

RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase delta

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ~10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.