Template switching in DNA replication can create and maintain RNA hairpins.

The evolutionary origin of RNA stem structures and the preservation of their base-pairing under a spontaneous and random mutation process have puzzled theoretical evolutionary biologists.

  • DNA replication-related template switching is a mutation mechanism that creates reverse-complement copies of sequence regions within a genome by replicating briefly along either the complementary or nascent DNA strand.
  • Depending on the relative positions and context of the four switch points, this process may produce a reverse-complement repeat capable of forming the stem of a perfect DNA hairpin or fix the base pairing of an existing stem. Template switching is typically thought to trigger large structural changes, and its possible role in the origin and evolution of RNA genes has not been studied.
  • Here, we show that the reconstructed ancestral histories of RNA genes contain mutation patterns consistent with the DNA replication-related template switching.
  • In addition to multibase compensatory mutations, the mechanism can explain complex sequence changes, although mutations breaking the structure rarely get fixed in evolution.
  • Our results suggest a solution for the long-standing dilemma of RNA gene evolution and demonstrate how template switching can both create perfect stems with a single mutation event and help maintaining the stem structure over time nucleotides embedded in template DNA impair mitochondrial RNA polymerase progression
  • Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome.
  • Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed.
  • Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM.
  • Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.

Programmed Assembly of DNA Templates by Silver Nanowires.

DNA origami templates are known to exhibit many advantages to integrate functional components at desirable locations for nanoelectronic applications.

In order to immobilize conducting or semiconducting species in a bottom-up approach, the programmed assembly of DNA templates is of utmost necessity.

This report demonstrates the silver nanowires enabled bridging of two linear DNA origami (DO) nanostructures by utilizing the host-guest interaction of biotin-STV and sequence-specific silver metallization of poly(dG-dC) DNA nanowires (in 10 % yield) using (dA)10 coated AgNPs (15 nm).

The enzymatic synthesis of 750 bp, 1500 bp and 3000 bp bis-biotinylated poly(dG-dC), facile synthesis of 1 : 1 biotin-STV and silver-nanowire bridged DNA templates were characterized by gel electrophoresis, atomic force microscope imaging techniques. The strategy utilized here provides a method that can precisely connect heterogeneous templates towards bottom-up fabrication of practical nanoelectronics.

Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production.

  1. Performance variability is a common challenge in cell-free protein production and hinders a wider adoption of these systems for both research and biomanufacturing.
  2. While the inherent stochasticity and complexity of biology likely contributes to variability, other systematic factors may also play a role, including the source and preparation of the cell extract, the composition of the supplemental reaction buffer, the facility at which experiments are conducted, and the human operator (Cole et al. ACS Synth Biol 8:2080-2091, 2019).
  3. Variability in protein production could also arise from differences in the DNA template-specifically the amount of functional DNA added to a cell-free reaction and the quality of the DNA preparation in terms of contaminants and strand breakage. Here, we present protocols and suggest best practices optimized for DNA template preparation and quantitation for cell-free systems toward reducing variability in cell-free protein production.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template.

For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase.

Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis.

We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT.

Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Quick and sensitive colorimetric detection of amino acid with functionalized-silver/copper nanoparticles in the presence of cross linker, and bacteria detection by using DNA-template nanoparticles as peroxidase activity

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In this project, poly (citric acid) (PCA) functionalized on nano Ag/Cu was synthesized by chemical analysis method. The nano probe was applied to detection of cysteine by using the magnesium (II) ions as a cross linker. The characterization of Ag/Cu/PCA nano probe was studied by using the UV-visible, morphological microscopy, dynamic light scattering, and zeta potential analyzer.

The zeta potential and size of Ag/Cu/PCA were -38.0 mV and 18.0 nm, respectively. The prepared nano probe shows rapid response for detection of cysteine.

The detection limit of Ag/Cu/PCA nano probe was 0.07 nM. Additional, the Ag/Cu/PCA nanoparticles was applied to cysteine detection from real samples in the presence of amino acids compounds.

Rapidly and sensitive determination of Streptococcus pneumoniae is substantial for food safety and human health. The DNA-Ag/Cu/PCA were prepared as a template in chemical method and experimented as a bio-receptor for the cell bacteria detection as peroxidase-like catalytic process. The DNA-Ag/Cu/PCA nano probe shows a linear dynamic concertation range of Streptococcus pneumoniae via detection limit about 65 CFU/mL. The project presents that the DNA-Ag/Cu/PCA could detect the biological and bacterial samples via high accuracy.

Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA.

The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the ‘replication fork trap’ region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites.

Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface.

To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro.

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pBR322 DNA

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pUC18 DNA

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DNA-fectamine

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We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication.

Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

 

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