scholarly journals Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

2021 ◽  
Author(s):  
Inyup Paik ◽  
Phuoc H. T. Ngo ◽  
Raghav Shroff ◽  
Andre C. Maranhao ◽  
David J.F. Walker ◽  
...  
Keyword(s):  
Machine Learning ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Lamp Assay ◽  
Enzyme Engineering ◽  
Amino Acid Substitutions ◽  
Geobacillus Stearothermophilus ◽  
Transcription Activity ◽  
Rational Engineering ◽  
Engineering Strategy

ABSTRACTDNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 °C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

A systematic exploration of ΔΔG cutoff ranges in machine learning models for protein mutation stability prediction

2018 ◽  
Vol 16 (05) ◽  
pp. 1840022 ◽  
Author(s):  
Richard Olney ◽  
Aaron Tuor ◽  
Filip Jagodzinski ◽  
Brian Hutchinson
Keyword(s):  
Machine Learning ◽  
Amino Acid ◽  
Amino Acid Substitutions ◽  
Learning Models ◽  
Systematic Exploration ◽  
Wide Range ◽  
The Stability ◽  
Experimental Values ◽  
Machine Learning Models

Discerning how a mutation affects the stability of a protein is central to the study of a wide range of diseases. Mutagenesis experiments on physical proteins provide precise insights about the effects of amino acid substitutions, but such studies are time and cost prohibitive. Computational approaches for informing experimentalists where to allocate wet-lab resources are available, including a variety of machine learning models. Assessing the accuracy of machine learning models for predicting the effects of mutations is dependent on experiments for amino acid substitutions performed in vitro. When similar experiments on physical proteins have been performed by multiple laboratories, the use of the data near the juncture of stabilizing and destabilizing mutations is questionable. In this work, we explore a systematic and principled alternative to discarding experimental data close to the juncture of stabilizing and destabilizing mutations. We model the inconclusive range of experimental [Formula: see text] values via 3- and 5-way classifiers, and systematically explore potential boundaries for the range of inconclusive experimental values. We demonstrate the effectiveness of potential boundaries through confusion matrices and heat map visualizations. We explore two novel metrics for assessing viable cutoff ranges, and find that under these metrics, a lower cutoff near [Formula: see text] and an upper cutoff near [Formula: see text] are optimal across multiple machine learning models.

scholarly journals Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

2003 ◽  
Vol 278 (21) ◽  
pp. 19341-19346 ◽  
Author(s):  
Eitan Glick ◽  
Janice S. Chau ◽  
Kellie L. Vigna ◽  
Scott D. McCulloch ◽  
Elinor T. Adman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Amino Acid Substitutions ◽  
Human Dna ◽  
Conserved Tyrosine

scholarly journals Possible changes in fidelity of DNA polymerase δ in ancestral mammals

2020 ◽  
Author(s):  
Kazutaka Katoh ◽  
Naoyuki Iwabe ◽  
Takashi Miyata
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Phylogenetic Analyses ◽  
Amino Acid Level ◽  
Amino Acid Substitutions ◽  
Dna Polymerase Δ ◽  
Time Period ◽  
Mammalian Lineage ◽  
Exonuclease Domain

AbstractDNA polymerase δ (polδ) is one of the major DNA polymerases that replicate chromosomal genomes in eukaryotes. Given the essential role of this protein, its phylogenetic tree was expected to reflect the relationship between taxa, like many other essential proteins. However, the tree of the catalytic subunit of polδ showed an unexpectedly strong heterogeneity among vertebrate lineages in evolutionary rate at the amino acid level, suggesting unusual amino acid substitutions specifically in the ancestral mammalian lineage. Structural and phylogenetic analyses were used to pinpoint where and when these amino acid substitutions occurred: around the 3′-5′ exonuclease domain in later mammal ancestry, after the split between monotremes and therians. The 3′-5′ exonuclease domain of this protein is known to have an impact on the fidelity of replication. Based on these observations, we explored the possibility that the amino acid substitutions we identified in polδ affected the mutation rate of entire chromosomal genomes in this time period.

scholarly journals Machine learning integration for predicting the effect of single amino acid substitutions on protein stability

2009 ◽  
Vol 9 (1) ◽  
pp. 66 ◽  
Author(s):  
Ayşegül Özen ◽  
Mehmet Gönen ◽  
Ethem Alpaydın ◽  
Türkan Haliloğlu
Keyword(s):  
Machine Learning ◽  
Amino Acid ◽  
Protein Stability ◽  
Single Amino Acid ◽  
Amino Acid Substitutions

Plasmodium falciparum clones resistant to (S)-9-(3-hydroxy-2-phosphonylmethoxy-propyl)adenine carry amino acid substitutions in DNA polymerase δ

2000 ◽  
Vol 106 (1) ◽  
pp. 175-180 ◽  
Author(s):  
Louis J.J.W Smeijsters ◽  
Natasha M Zijlstra ◽  
Jetty Veenstra ◽  
Babs E Verstrepen ◽  
Carine Heuvel ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Amino Acid Substitutions ◽  
Dna Polymerase Δ

scholarly journals Identification of Escherichia coli dnaE(polC) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine

2000 ◽  
Vol 182 (14) ◽  
pp. 3942-3947 ◽  
Author(s):  
Koji Hiratsuka ◽  
Linda J. Reha-Krantz
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Protein Product ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Α Subunit ◽  
Nucleotide Interactions ◽  
Increased Sensitivity ◽  
Mutant Phenotypes

ABSTRACT Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679–680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE (polC) gene ofEscherichia coli that alter sensitivity to 2′,3′-dideoxyadenosine (ddA). Two dnaE (polC) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the α subunit, the protein product of the dnaE (polC) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative dnaE(polC) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the α subunit of the DNA polymerase III holoenzyme.

scholarly journals Normally lethal amino acid substitutions suppress an ultramutator DNA Polymerase δ variant

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Daniel G. Dennis ◽  
Jill McKay-Fleisch ◽  
Kaila Eitzen ◽  
Ian Dowsett ◽  
Scott R. Kennedy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Amino Acid Substitutions ◽  
Dna Polymerase Δ

scholarly journals Building better polymerases: Engineering the replication of expanded genetic alphabets

2020 ◽  
Vol 295 (50) ◽  
pp. 17046-17059
Author(s):  
Zahra Ouaray ◽  
Steven A. Benner ◽  
Millie M. Georgiadis ◽  
Nigel G. J. Richards
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Amino Acid Substitutions ◽  
Klenow Fragment ◽  
Base Pairs ◽  
Specific Amino Acid ◽  
Rational Engineering ◽  
Performance Specifications ◽  
Insight Into

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the “performance specifications” needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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