scholarly journals DNA polymerase activity on synthetic N3′→P5′ phosphoramidate DNA templates

2019 ◽  
Vol 47 (17) ◽  
pp. 8941-8949 ◽  
Author(s):  
Victor S Lelyveld ◽  
Derek K O’Flaherty ◽  
Lijun Zhou ◽  
Enver Cagri Izgu ◽  
Jack W Szostak
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Chemical Space ◽  
Genetic Material ◽  
Polymerase Activity ◽  
Dna Duplexes ◽  
Dna Templates ◽  
Reverse Transcriptases ◽  
The Universe ◽  
Rna And Dna

Abstract Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3′→P5′ phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

1. Microbial diversity

2014 ◽  
Author(s):  
Nicholas P. Money
Keyword(s):  
Microbial Diversity ◽  
Dna Sequences ◽  
Genetic Material ◽  
Genetic Identification ◽  
Single Chromosome ◽  
Gram Staining ◽  
Microbial P ◽  
Staining Techniques ◽  
Primary Groups ◽  
Rna And Dna

‘Microbial diversity’ considers the vast array of microorganisms—the smallest forms of life—which exist everywhere. The three primary groups of microorganisms are bacteria, archaea, and eukaryotes. Bacteria and archaea are prokaryotes with their genetic material held in a single chromosome. In eukaryotes, most of the genome is held in multiple chromosomes. Over 11,000 species of bacteria have been identified using microscopic identification of cell shape and metabolic activity, Gram-staining techniques, and genetic identification of RNA and DNA sequences. There are 500 named species of archaea, divided into two phyla: the euryarchaeota and the crenarchaeota. There are eight supergroupings of eukaryotes, all of them include single-celled organisms, and five are entirely microbial.

scholarly journals Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

2020 ◽  
Vol 117 (13) ◽  
pp. 7276-7283 ◽  
Author(s):  
Victor S. Lelyveld ◽  
Wen Zhang ◽  
Jack W. Szostak
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Bond Formation ◽  
Genetic Material ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Chain Elongation ◽  
Hydroxyl Group ◽  
Common Mechanism ◽  
Modified Dna

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase

1991 ◽  
Vol 11 (3) ◽  
pp. 1696-1706
Author(s):  
U Schulte ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Plasmid Dna ◽  
Sequence Similarity ◽  
Polymerase Activity ◽  
Open Reading Frame ◽  
Mitochondrial Plasmid ◽  
Reading Frame ◽  
Reverse Transcriptases ◽  
Neurospora Intermedia

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

scholarly journals Rabbit Endometrial RNA- and DNA-Dependent DNA Polymerase Activity

1978 ◽  
Vol 18 (3) ◽  
pp. 371-378 ◽  
Author(s):  
Beverly S. Chilton ◽  
J. C. Daniel
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Rna And Dna

scholarly journals The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase.

1991 ◽  
Vol 11 (3) ◽  
pp. 1696-1706 ◽  
Author(s):  
U Schulte ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Plasmid Dna ◽  
Sequence Similarity ◽  
Polymerase Activity ◽  
Open Reading Frame ◽  
Mitochondrial Plasmid ◽  
Reading Frame ◽  
Reverse Transcriptases ◽  
Neurospora Intermedia

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

Sign in / Sign up

Export Citation Format

Share Document