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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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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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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
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Studying DNA Looping by Single-Molecule FRET
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Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.

Kevin D P Aris1,2, Joshua C Cofsky3,4, Honglue Shi5,6

  • 1Biophysics Program, Stanford University, Stanford, CA, USA.

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|March 26, 2025
PubMed
Summary
This summary is machine-generated.

CRISPR-Cas12a

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetic Engineering

Background:

  • CRISPR-Cas12a is a versatile tool in genetic engineering due to its programmable DNA binding and cleavage.
  • Cas12a functions as an RNA-guided endonuclease, forming a three-stranded R-loop structure for DNA target engagement.

Purpose of the Study:

  • To investigate the dynamics and mechanics of R-loop formation in CRISPR-Cas12a.
  • To resolve R-loop formation at base-pair resolution using single-molecule torque spectroscopy.

Main Methods:

  • Single-molecule torque spectroscopy
  • Analysis of CRISPR-Cas12a orthologs
  • Base-pair resolution kinetic studies

Main Results:

  • Direct observation of kinetic intermediates (~5 bp and ~17 bp) during R-loop formation.
  • Identification of transient DNA unwinding beyond the 20 bp R-loop.
  • R-loop formation landscape is dependent on Cas12a ortholog, target sequence, mismatches, and DNA supercoiling.

Conclusions:

  • A four-state kinetic model accurately describes Cas12a R-loop dynamics.
  • Provides a biophysical framework for understanding Cas12a activity and specificity.
  • Highlights the complex, multi-state nature of R-loop formation.