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Related Concept Videos

Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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...
Nucleic Acid Structure01:25

Nucleic Acid Structure

The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
DNA Structure
DNA has a double-helix structure. The...
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Homologous Recombination02:31

Homologous Recombination

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...
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
DNA Helicases00:55

DNA Helicases

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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DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition
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Structure basis for single-strand nucleic acid targeting by IscB and variants.

Chengtao Xu1, Qi Yang1, Xiaolin Niu1

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, United States.

Nucleic Acids Research
|June 22, 2026
PubMed
Summary

The study reveals how IscB, an ancestor of CRISPR-Cas9, binds to single-stranded nucleic acids (ssNA) using a unique roadblock mechanism. Mutations enhancing this process improved RNA editing efficiency in human cells.

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Gene Editing

Background:

  • CRISPR-Cas9 technology is derived from the transposon-encoded IscB.
  • IscB and related Cas9 enzymes have been engineered into RNA editors by modifying their DNA recognition domains.
  • Understanding the mechanism of IscB is crucial for advancing RNA editing tools.

Purpose of the Study:

  • To elucidate the structural basis of IscB's interaction with single-stranded nucleic acid (ssNA) targets.
  • To understand the mechanism of RNA targeting by IscB, with or without its target-adjacent motif (TAM)-interacting domain (TID).
  • To engineer improved IscB variants for enhanced RNA editing efficiency.

Main Methods:

  • Four cryo-electron microscopy (cryo-EM) structures of IscB were determined in complex with ssNA targets.
  • Structural analysis was performed to understand IscB-ssNA interactions and conformational changes.
  • Mutagenesis studies were conducted to alter IscB's ssNA binding and nuclease activity.

Main Results:

  • IscB engages ssNA through a conserved mechanism involving initial seed duplex formation and a roadblock by the HNH nuclease.
  • Full duplex formation is required to dislodge the HNH roadblock, exposing the nuclease active sites.
  • Mutations improving ssNA binding or HNH dislodging enhanced IscB's RNA-targeting efficiency in vitro and in human cells.

Conclusions:

  • The structural and mechanistic insights into IscB-ssNA interactions provide a foundation for developing improved RNA editing technologies.
  • Engineered IscB variants demonstrate enhanced RNA editing capabilities, paving the way for more precise gene editing applications.
  • This research deepens our understanding of RNA-guided endonucleases and their potential in biotechnology.