All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 °C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445062 | PMC |
| http://dx.doi.org/10.1038/srep10404 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Genomic analysis and replication kinetics of the closely related EHV-1 neuropathogenic 21P40 and abortigenic 97P70 strains.
Vet Res
January 2025
Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, 9820, Merelbeke, Belgium.
Varicellovirus equidalpha 1, formerly known as Equid alphaherpesvirus 1 (EHV-1), is highly prevalent and can lead to various problems, such as respiratory problems, abortion, neonatal foal death, and neurological disorders. The latter is known as equine herpes myeloencephalopathy (EHM). Cases of EHM have significantly increased since the beginning of the twenty-first century.
View Article and Find Full Text PDFTranscriptional dynamics during Heliothis zea nudivirus 1 infection in an ovarian cell line from .
J Gen Virol
January 2025
Laboratory of Virology, Wageningen University and Research, 6708 PB Wageningen, Netherlands.
Nudiviruses (family ) are double-stranded DNA viruses that infect various insects and crustaceans. Among them, Heliothis zea nudivirus 1 (HzNV-1) represents the rare case of a lepidopteran nudivirus inducing a sexual pathology. Studies about molecular pathological dynamics of HzNV-1 or other nudiviruses are scarce.
View Article and Find Full Text PDFModulation of Cell Cycle Kinases by Kaposi's Sarcoma-Associated Herpesvirus.
J Med Virol
January 2025
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
The cell cycle is governed by kinase activity that coordinates progression through a series of regulatory checkpoints, preventing the division of damaged cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple genes that modulate or co-opt the activity of these kinases, shaping the cellular environment to promote viral persistence. By advancing the cell cycle, KSHV facilitates latent replication and subsequent transmission of viral genomes to daughter cells, while also contributing to the establishment of multiple cancer types.
View Article and Find Full Text PDFChronically persistent viruses are integral components of the organismal ecosystem in humans and animals . Many of these viruses replicate and accumulate within the cell nucleus . The nuclear location allows viruses to evade cytoplasmic host viral sensors and promotes viral replication .
View Article and Find Full Text PDFChanges in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes in many organisms. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs that are present in only a few genomes across a population are often overlooked but important; if beneficial under specific conditions, a de novo CNV that arises in a single genome can expand during selection to create a larger population of cells with novel characteristics.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!