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DNA Topoisomerases02:02

DNA Topoisomerases

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Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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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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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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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,...
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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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Stepwise DNA unwinding gates TnpB genome-editing activity.

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Researchers improved genome engineering by stabilizing DNA unwinding states in TnpB enzymes. This enhances DNA cleavage and editing efficiency, overcoming limitations of natural TnpB (transposase B) systems.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • TnpB (transposase B) is an RNA-guided endonuclease and a precursor to CRISPR-Cas12, holding potential for genome engineering.
  • The genome-editing capabilities of TnpBs are currently limited, with the factors influencing their activity not well understood.

Purpose of the Study:

  • To investigate the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB).
  • To identify strategies for enhancing TnpB activity for improved genome engineering applications.

Main Methods:

  • Biochemical assays were employed to analyze the DNA-unwinding process.
  • Single-molecule assays were utilized to observe the dynamic states of DNA unwinding by Ymu1 TnpB.

Main Results:

  • DNA unwinding by Ymu1 TnpB involves intermediate and fully unwound states, with the latter being unstable without negative supercoiling.
  • An optimized variant, Ymu1-WFR, was developed that stabilizes these unwinding states.
  • Ymu1-WFR demonstrated enhanced DNA cleavage in vitro and increased genome editing efficiency in vivo.

Conclusions:

  • The study elucidates the physical basis for the limited activity of natural TnpBs.
  • Stabilizing specific DNA unwinding states is key to enhancing TnpB-mediated DNA targeting and genome editing efficacy.