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Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...
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Homologous Recombination

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Protein-Primed DNA Replication is Restored in a Modified Cell-Free Protein Synthesis System.

Riku Nagai1, Carlos Chavez Ramirez1, Zhanar Abil1,2,3

  • 1Department of Biology, University of Florida, Gainesville, Florida 32611, United States.

ACS Synthetic Biology
|May 19, 2026
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Summary
This summary is machine-generated.

Researchers improved gene evolution by enhancing DNA amplification in synthetic microcompartments. This optimization boosts the efficiency of in vitro directed evolution (IVDE) for developing novel gene functions.

Keywords:
cell-free directed evolutioncell-free gene expressionclonal DNA amplificationin vitro evolutionin vitro transcription-translation (IVTT)in vitro transcription−translation−coupled DNA replication (IVTTR)

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biotechnology

Background:

  • In vitro directed evolution (IVDE) in synthetic microcompartments enables gene function evolution beyond simple affinity.
  • A key challenge is ensuring single DNA template molecules per microcompartment for genotype-phenotype linkage, which often leads to inefficient in vitro transcription and translation (IVTT) and poor DNA recovery.
  • Previous development of Clonal Amplification-enhanceD Gene Expression (CADGE) aimed to resolve this by enabling clonal DNA amplification and in situ transcription-translation within Protein synthesis Using Recombinant Elements (PURE) systems.

Purpose of the Study:

  • To address the limitations of current commercial PURE systems in supporting DNA replication for the CADGE strategy.
  • To restore DNA amplification levels in CADGE to previously reported efficiencies.
  • To optimize IVDE protocols for enhanced gene evolution.

Main Methods:

  • Utilized Protein synthesis Using Recombinant Elements (PURE) systems for gene expression.
  • Implemented the Clonal Amplification-enhanceD Gene Expression (CADGE) strategy for clonal DNA amplification and in situ transcription-translation.
  • Replaced commercial energy mixes in PURE systems with homemade formulations known to enhance rolling circle amplification.

Main Results:

  • Observed significantly lower DNA replication in commercial PURE systems than previously reported.
  • Successfully restored DNA amplification in CADGE to previously achieved levels by using homemade energy mixes.
  • Demonstrated enhanced performance of the CADGE strategy with optimized PURE system components.

Conclusions:

  • Homemade energy mixes can significantly improve DNA amplification in PURE systems for the CADGE strategy.
  • Optimized PURE systems enhance the efficiency of IVDE by improving DNA replication and gene expression.
  • This work advances the application of synthetic microcompartments for directed evolution of gene functions.