Wild-type Polymerase Research Articles

DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants.

The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines at the end of DNA templates enhance the DNA-terminus-initiated transcription. Moreover, we found that aromatic residues located at position 47 in the C-helixleadto a significant reduction in the production of full-length dsRNA. As a result, the mRNA synthesized using the T7 RNA polymerase G47W mutant exhibits higher expression efficiency and lower immunogenicity compared to the mRNA produced using the wild-type T7 RNA polymerase.

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.

Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature. To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used. The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis.

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization

Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins.

A simple bypass assay for DNA polymerases shows that cancer-associated hypermutating variants exhibit differences in vitro.

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease-defective and two hypermutating human DNA polymerase ε variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild-type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed misincorporation rate comparable to the exonuclease dead enzyme rather than P286R, suggesting that there may be some differences in the way that these variants cause hypermutation. Using this assay, misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C-to-T transitions. This is consistent with the observation that C-to-T transitions are commonly seen in DNA polymerase ε mutant tumours.

Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing.

A model for the catalytic activity of microtubule polymerases.

A XMAP215/Stu2/Alp14 polymerase can catalyze processively the tubulin addition to the microtubule (MT) plus end. In this work, a model is proposed for the underlying molecular mechanism of the polymerase activity, where the polymerase can not only catalyze processively the tubulin addition to but also promote the tubulin removal from the MT plus end. Based on the model the dynamics of both the wild-type and mutant polymerases is studied theoretically, explaining consistently and well various available experimental data. To further test the model, predicted results are provided.

Global genomic instability caused by reduced expression of DNA polymerase ε in yeast

SignificanceAlthough most studies of the genetic regulation of genome stability involve an analysis of mutations within the coding sequences of genes required for DNA replication or DNA repair, recent studies in yeast show that reduced levels of wild-type enzymes can also produce a mutator phenotype. By whole-genome sequencing and other methods, we find that reduced levels of the wild-type DNA polymerase ε in yeast greatly increase the rates of mitotic recombination, aneuploidy, and single-base mutations. The observed pattern of genome instability is different from those observed in yeast strains with reduced levels of the other replicative DNA polymerases, Pol α and Pol δ. These observations are relevant to our understanding of cancer and other diseases associated with genetic instability.

Heat Treatment Promotes Ubiquitin-Mediated Proteolysis of SARS-CoV-2 RNA Polymerase and Decreases Viral Load

Despite extensive efforts, COVID-19 pandemic caused by the SARS-CoV-2 virus is still at large. Vaccination is an effective approach to curb virus spread, but several variants (e.g., delta, delta plus, omicron, and IHU) appear to weaken or possibly escape immune protection. Thus, novel and quickly scalable approaches to restrain SARS-CoV-2 are urgently needed. Multiple evidences showed thermal sensitivity of SARS-CoV-2 and negative correlation between environmental temperature and COVID-19 transmission with unknown mechanism. Here, we reveal a potential mechanism by which mild heat treatment destabilizes the wild-type RNA-dependent RNA polymerase (also known as nonstructural protein 12 (NSP12)) of SARS-CoV-2 as well as the P323L mutant commonly found in SARS-CoV-2 variants, including omicron and IHU. Mechanistically, heat treatment promotes E3 ubiquitin ligase ZNF598-dependent NSP12 ubiquitination leading to proteasomal degradation and significantly decreases SARS-CoV-2 RNA copy number and viral titer. A mild daily heat treatment maintains low levels of both wild-type and P323L mutant of NSP12, suggesting clinical potential. Collectively, this novel mechanism, heat-induced NSP12 degradation, suggests a prospective heat-based intervention against SARS-CoV-2.

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases.

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.

Abstract 808: Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer

Abstract Background: Early-onset renal cancer (eoRC) is typically associated with significant family history of cancer, and often associated with germline pathogenic variants (PVs) in ‘RC-specific' genes including VHL, MET, FLCN, TSC1, TSC2, FH, SDH, PTEN and BAP1. However, most eoRC patients lack PVs in RC-specific genes; rather, we recently identified an enrichment of likely PVs in DNA repair genes in eoRC patients. Here, using eoRC patient resources (primary and transformed lymphocytes, lymphocyte DNA, and tumor tissue), we functionally evaluated the DNA repair variants identified in eoRC patients. Methods: We performed whole-exome sequencing (WES) on lymphocyte DNA from 22 patients with genetically undefined eoRC, with extensive family history of RC and/or other cancers, provided by Risk Assessment Program and Genitourinary Group at Fox Chase Cancer Center. Prioritized variants were assessed by structural, biochemical and cellular studies. Results: 70% of patients had rare, heterozygous, missense variants in genes that suppress DNA damage. ~18% of the variants were in DNA polymerase genes (POLD1, POLE, POLH, POLK), which mediate DNA replication-repair. Increased tumor mutation burden (>10 mutations/Mb) was observed in tumors carrying predicted damaging polymerase variants. In functional testing, induction of DNA damage in primary lymphocytes from eoRC patients and matched cancer-free controls (n=20 each) selectively elevated γH2AX foci (indicative of DNA damage) in cells from eoRC patients (P<0.001, pairwise T-test). DNA fiber analysis of eoRC patient-derived lymphocyte lines carrying the polymerase variants, showed significantly slower rate of DNA replication, and replication fork restoration after DNA damage versus the matched control-derived lymphocyte lines (P<0.01, Mann-Whitney test). Biochemical analysis of purified polymerase proteins found that the variants differ from wild type polymerase in their ability to extend DNA substrates and bypass DNA lesion(s), and exhibit dominant negative effects. Conclusion and future work: Our results indicate that germline variants in DNA repair genes impact suppression of DNA damage in cells from patients with eoRC. Biological studies testing the impact of the identified variants on renal carcinogenesis are warranted. Overall, genetic testing for variation in DNA repair genes may provide a more comprehensive risk assessment in eoRC patients, and provide novel opportunities for targeting DNA repair vulnerabilities in RC. Citation Format: Elena V. Demidova, Waleed Iqbal, Ilya G. Serebriiskii, Andrea F. Forman, Tatiana Kent, Richard T. Pomerantz, Erica A. Golemis, Michael J. Hall, David Y. Chen, Mary B. Daly, Sanjeevani Arora. Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 808.

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

Evaluation of repair activity by quantification of ribonucleotides in the genome

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ‐seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ‐seq analysis of cells harboring wild‐type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3′‐shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild‐type and repair‐deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ‐seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ‐seq can provide an estimate of indel risk.

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

An assay for DNA polymerase β lyase inhibitors that engage the catalytic nucleophile for binding

DNA polymerase β (Pol β) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol β thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase β (Pol β) and Pol β modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

Detection of TERT promoter mutation in serum cell-free DNA using wild-type blocking PCR combined with Sanger sequencing in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promoter mutation is the most frequent genetic alteration in hepatocellular carcinoma (HCC). However, there is currently no suitable highly sensitive method that can detect such mutation using serum cell-free DNA (cfDNA). We analyzed somatic point mutations that substitute cytosine for thymidine at position 228 (C228T), as one of the hotspots of TERT promoter mutations, in serum cfDNA using a highly sensitive detection method of wild-type blocking polymerase chain reaction (WTB-PCR) combined with Sanger sequencing. In TERT promoter mutation sensitivity study, synthetic oligonucleotides were prepared to determine the lowest detection limit of the WTB-PCR, using serial dilutions of mutant-type (MT) DNA in the background of wild-type (WT) DNA. Using this technique, we conducted a longitudinal study in one patient who developed HCC during the follow-up and determined the relationship between HCC and TERT C228T in serum cfDNA. In the sensitivity study, the mutant peak at position 228 was detected at 0.7% or higher but was not detected at 0.6%. Thus, sequencing analysis of WTB-PCR product demonstrated the limit of detection in excess of 0.7% MT DNA in the background of WT DNA. One male patient with HCV-related cirrhosis developed HCC during the follow-up. TERT C228T was negative before the diagnosis of HCC, positive at the diagnosis of HCC and did not increase with advancement of malignancy. We developed a highly sensitive method for detection of TERT promoter mutation using WTB-PCR combined with Sanger sequencing and demonstrated its clinical usefulness in the measurement of TERT C228T in serum cfDNA. Larger studies are needed to confirm these results and establish the clinical utility of this new method.

Uncovering the Resistance Mechanism of Mycobacterium tuberculosis to Rifampicin Due to RNA Polymerase H451D/Y/R Mutations From Computational Perspective.

Tuberculosis is still one of the top 10 causes of deaths worldwide, especially with the emergence of multidrug-resistant tuberculosis. Rifampicin, as the most effective first-line antituberculosis drug, also develops resistance due to the mutation on Mycobacterium tuberculosis (Mtb) RNA polymerase. Among these mutations, three mutations at position 451 (H451D, H451Y, H451R) are associated with high-level resistance to rifampicin. However, the resistance mechanism of Mtb to rifampicin is still unclear. In this work, to explore the resistance mechanism of Mtb to rifampicin due to H451D/Y/R mutations, we combined the molecular dynamics simulation, molecular mechanics generalized-Born surface area calculation, dynamic network analysis, and residue interactions network analysis to compare the interaction change of rifampicin with wild-type RNA polymerase and three mutants. The results of molecular mechanics generalized-Born surface area calculations indicate that the binding free energy of rifampicin with three mutants decreases. In addition, the dynamic network analysis and residue interaction network analysis show that when H451 was mutated, the interactions of residue 451 with its adjacent residues such as Q438, F439, M440, D441, and S447 disappeared or weakened, increasing the flexibility of binding pocket. At the same time, the disappearance of hydrogen bonds between R613 and rifampicin caused by the flipping of R613 is another important reason for the reduction of binding ability of rifampicin in H451D/Y mutants. In H451R mutant, the mutation causes the binding pocket change too much so that the position of rifampicin has a large movement in the binding pocket. In this study, the resistance mechanism of rifampicin at the atomic level is proposed. The proposed drug-resistance mechanism will provide the valuable guidance for the design of antituberculosis drugs.

The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion.

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

A Molecular Dynamics Investigation of the Thermostability of Cold-Sensitive I707L KlenTaq1 DNA Polymerase and Its Wild-Type Counterpart.

DNA polymerase I from Thermus aquaticus ( Taq DNA polymerase) is useful for polymerase chain reactions because of its exceptional thermostability; however, its activity at low temperatures can cause amplification of unintended products. Mutation of isoleucine 707 to leucine (I707L) slows Taq DNA polymerase at low temperatures, which decreases unwanted amplification due to mispriming. In this work, unrestrained molecular dynamics (MD) simulations were performed on I707L and wild-type (WT) Taq DNA polymerase at 341 and 298 K to determine how the mutation affects the dynamic nature of the protein. The results suggest that I707L Taq DNA polymerase remains relatively immobile at room temperature and becomes more flexible at the higher temperature, while the WT Taq DNA polymerase demonstrates less substantial differences in dynamics at high and low temperatures. These results are in agreement with previous experimental results on the I707L mutant Taq DNA polymerase that show dynamic differences at high and low temperatures. The decreased mobility of the mutant at low temperature suggests that the mutant remains longer in the blocked conformation, and this may lead to reduced activity relative to the WT at 298 K. Principal component analysis revealed that the mutation results in decoupled movements of the Q helix and fingers domain. This decoupled nature of the mutant gives way to an increasingly flexible N-terminal end of the Q helix at 341 K, a characteristic not seen for WT Taq DNA polymerase.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.