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Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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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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Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome.  Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form...
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In eukaryotic DNA replication, a single-stranded DNA fragment remains at the end of a chromosome after the removal of the final primer. This section of DNA cannot be replicated in the same manner as the rest of the strand because there is no 3’ end to which the newly synthesized DNA can attach. This non-replicated fragment results in gradual loss of the chromosomal DNA during each cell duplication. Additionally, it can induce a DNA damage response by enzymes that recognize single-stranded...
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Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs
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Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.

Basil P Hubbard1, Ahmed H Badran1, John A Zuris1

  • 1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

Nature Methods
|August 11, 2015
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Summary

We developed DNA-binding phage-assisted continuous evolution (DB-PACE) to improve genome editing tools. This method enhances the specificity of DNA-binding nucleases like TALENs for safer gene therapies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Programmable nucleases with DNA-binding domains are crucial for genome alteration in model organisms.
  • These nucleases hold significant promise for developing novel human therapeutics.
  • Enhancing the specificity of these agents is critical for safe and effective therapeutic applications.

Purpose of the Study:

  • To introduce DNA-binding phage-assisted continuous evolution (DB-PACE) as a general laboratory evolution method.
  • To evolve DNA-binding activity and specificity of nuclease-based genome editing tools.
  • To improve the accuracy and safety of genome editing agents.

Main Methods:

  • Development and application of the DB-PACE system for protein evolution.
  • Laboratory evolution of transcription activator-like effectors nucleases (TALENs) using DB-PACE.
  • Assessment of DNA cleavage specificity of evolved TALENs.

Main Results:

  • DB-PACE was established as a versatile platform for evolving DNA-binding proteins.
  • TALENs with significantly improved DNA cleavage specificity were generated.
  • Demonstrated the ability of DB-PACE to enhance the accuracy of genome-editing agents.

Conclusions:

  • DB-PACE is a powerful approach for laboratory evolution of DNA-binding activity and specificity.
  • The enhanced specificity of TALENs generated via DB-PACE has implications for safer genome editing.
  • DB-PACE represents a versatile strategy for improving the precision of gene-editing technologies for therapeutic use.