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Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507 -
Communications Chemistry Jun 2024Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our... (Review)
Review
Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.
PubMed: 38890393
DOI: 10.1038/s42004-024-01216-0 -
Biology Jul 2023The metabarcoding methods for studying the diversity of freshwater microalgae and routine biomonitoring are actively used in modern research. A lot of experience has... (Review)
Review
The metabarcoding methods for studying the diversity of freshwater microalgae and routine biomonitoring are actively used in modern research. A lot of experience has been accumulated already, and many methodological questions have been solved (such as the influence of the methods and time of sample conservation, DNA extraction and bioinformatical processing). The reproducibility of the method has been tested and confirmed. However, one of the main problems-choosing a genetic marker for the study-still lacks a clear answer. We analyzed 70 publications and found out that studies on eukaryotic freshwater microalgae use 12 markers (different nuclear regions 18S and ITS and plastids L, 23S and 16S). Each marker has its peculiarities; they amplify differently and have various levels of efficiency (variability) in different groups of algae. The V4 and V9 18S and L regions are used most often. We concentrated especially on the studies that compare the results of using different markers and microscopy. We summarize the data on the primers for each region and on how the choice of a marker affects the taxonomic composition of a community.
PubMed: 37508467
DOI: 10.3390/biology12071038 -
Nature Structural & Molecular Biology Oct 2023DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the...
DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.
Topics: DNA Polymerase III; DNA; DNA Replication; RNA; DNA Ligases; DNA Ligase ATP; Endonucleases
PubMed: 37620586
DOI: 10.1038/s41594-023-01071-y -
The Journal of Parasitology Oct 2023Conventional PCR provides Leishmania species characterization with even a small amount of biological material. Species-specific primers have been a widely used...
Conventional PCR provides Leishmania species characterization with even a small amount of biological material. Species-specific primers have been a widely used alternative; however, nonspecific amplifications are a reality, interfering with PCR efficiency. In endemic areas with multiple etiological agents for leishmaniasis, there is a requirement for higher specificity of primers. This study evaluates 3 pairs of primers described for the identification and characterization of Leishmania infantum. Primers RV1/RV2, LEISH1/LEISH2, and FLC2/RLC2 were used with the DNA of L. infantum, Leishmania amazonensis, and Leishmania braziliensis. An initial temperature curve was performed (52-62 C) to determine the optimal annealing temperature, followed by a dilution curve of Leishmania DNA (500 pg/μl, 50 pg/μl, 5 pg/μl, 500 fg/μl, 50 fg/μl, 5 fg/μl, and 0.5 fg/μl) to be used for analytical sensitivity. RV1/RV2 PCR amplified L. infantum and L. amazonensis at all analyzed temperatures; LEISH1/LEISH2 PCR amplified all 3 species of Leishmania, although at some temperatures L. infantum was specifically amplified, and, finally, FLC2/RLC2 PCR amplified only L. infantum at all temperatures analyzed. In terms of sensitivity, RV1/RV2 PCR detected 1 fg of L. infantum DNA and 100 pg of L. amazonensis DNA; LEISH1/LEISH2 PCR detected 1 fg of L. infantum DNA, 100 fg of L. amazonensis DNA, and 10 fg of L. braziliensis DNA; and FLC2/RLC2 PCR detected 10 fg of L. infantum DNA. Thus, PCR with FLC2/RLC2 primers is best suited for the molecular characterization of L. infantum, especially in areas where there is an incidence of more than 1 Leishmania species, such as South America.
Topics: Leishmania infantum; Polymerase Chain Reaction; Leishmania mexicana; South America; Species Specificity
PubMed: 37668295
DOI: 10.1645/21-130 -
Molecular Ecology Resources May 2024Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and...
Surveying biodiversity has taken a quantum leap with environmental DNA (eDNA) metabarcoding, an immensely powerful approach lauded for its efficiency, sensitivity, and non-invasiveness. This approach emerges as a game-changer for the elusive realm of endangered and rare species-think nocturnal, environmentally elusive amphibians. Here, we have established a framework for constructing a reliable metabarcoding pipeline for amphibians, covering primer design, performance evaluation, laboratory validation, and field validation processes. The Am250 primer, located on the mitochondrial 16S gene, was optimal for the eDNA monitoring of amphibians, which demonstrated higher taxonomic resolution, smaller species amplification bias, and more extraordinary detection ability compared to the other primers tested. Am250 primer exhibit an 83.8% species amplification rate and 75.4% accurate species identification rate for Chinese amphibians in the in silico PCR and successfully amplified all tested species of the standard samples in the in vitro assay. Furthermore, the field-based mesocosm experiment showed that DNA can still be detected by metabarcoding even days to weeks after organisms have been removed from the mesocosm. Moreover, field mesocosm findings indicate that eDNA metabarcoding primers exhibit different read abundances, which can affect the relative biomass of species. Thus, appropriate primers should be screened and evaluated by three experimental approaches: in silico PCR simulation, target DNA amplification, and mesocosm eDNA validation. The selection of a single primer set or multiple primers' combination should be based on the monitoring groups to improve the species detection rate and the credibility of results.
Topics: Animals; DNA, Environmental; Biological Monitoring; DNA Barcoding, Taxonomic; Amphibians; Biodiversity; DNA; Environmental Monitoring; Ecosystem
PubMed: 38345249
DOI: 10.1111/1755-0998.13931 -
Biophysical Journal Jun 2024DNA primase is an iron sulfur enzyme in DNA replication responsible for synthesizing short RNA primers that serve as starting points for DNA synthesis. The role of the...
DNA primase is an iron sulfur enzyme in DNA replication responsible for synthesizing short RNA primers that serve as starting points for DNA synthesis. The role of the [4Fe-4S] cluster is not well determined. Here, we calculate the redox potential of the [4Fe-4S] with and without DNA/RNA using continuum electrostatics. In addition, we identify the structural changes coupled to the oxidation/reduction. Our calculations show that the DNA/RNA primer lowers the redox potential by 110 and 50 mV for the [4Fe-4S] and [4Fe-4S] states, respectively. The oxidation of the cluster is coupled to structural changes that significantly reduce the binding energies between the DNA and the nearby residues. The negative charges accumulated by the DNA and the RNA primers induce the oxidation of the [4Fe-4S] cluster. This in turn stimulates structural changes on the DNA-protein interface that significantly reduce the binding energies.
Topics: Oxidation-Reduction; DNA Primase; RNA; Iron-Sulfur Proteins; Protein Binding; DNA; Thermodynamics; Models, Molecular
PubMed: 38733082
DOI: 10.1016/j.bpj.2024.05.007 -
Journal of Visualized Experiments : JoVE May 2024A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for... (Comparative Study)
Comparative Study
A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.
Topics: Animals; DNA; Polymerase Chain Reaction; Calliphoridae
PubMed: 38767369
DOI: 10.3791/66737 -
Nature Communications Jul 2023The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is...
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
Topics: Bacteriophage T4; DNA Primase; DNA Helicases; DNA Replication; DNA Primers; DNA, Viral
PubMed: 37474605
DOI: 10.1038/s41467-023-40106-2 -
RNA Biology Jan 2024The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has...
The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.
Topics: DNA; RNA; RNA, Catalytic; Evolution, Molecular; RNA-Dependent RNA Polymerase; Artificial Cells
PubMed: 38785360
DOI: 10.1080/15476286.2024.2355391