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Related Concept Videos

Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
Homologous Recombination02:31

Homologous Recombination

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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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Identification of Functional Protein Regions Through Chimeric Protein Construction
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Published on: January 8, 2019

A chimeric Cre recombinase with regulated directionality.

David Warren1, Gurunathan Laxmikanthan, Arthur Landy

  • 1Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.

Proceedings of the National Academy of Sciences of the United States of America
|November 18, 2008
PubMed
Summary
This summary is machine-generated.

Engineered Cre recombinase gains regulated directionality and complex DNA target recombination by fusing with Int protein domain. This chimeric system mimics evolutionary pathways for diverse site-specific recombinases.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biochemistry

Background:

  • Site-specific recombination and transposition are key genomic rearrangement pathways across life.
  • Recombinases like Cre and lambda Int are vital tools in genetic engineering, differing in regulation and application.
  • Cre-mediated recombination is unregulated and bidirectional, while lambda Int is regulated and typically used in vitro.

Purpose of the Study:

  • To investigate if a chimeric Cre recombinase, fused with the Int N-terminal domain, can achieve regulated and complex DNA recombination.
  • To determine the requirements and regulatory mechanisms of this novel chimeric recombinase system.

Main Methods:

  • Construction of a chimeric Cre recombinase by fusing Cre with the N-terminal domain of lambda Int.
  • Testing the chimeric Cre's ability to recombine complex DNA targets with multiple protein-binding sites.
  • Analyzing the role of accessory proteins (IHF, Xis, Fis) in regulating the chimeric Cre's recombination directionality and efficiency.

Main Results:

  • The chimeric Cre successfully recombined complex DNA targets (>200 bp with 16 binding sites).
  • This recombination required the Integration Host Factor (IHF) protein.
  • Recombination was unidirectional and regulated by the relative levels of IHF, Xis, and Fis, with Xis controlling directionality.

Conclusions:

  • A simple chimeric construction can confer regulated directionality and complexity to recombination.
  • This chimeric system provides insights into the evolution of diverse site-specific recombinases.
  • The engineered recombinase offers a flexible tool for advanced genetic engineering applications.