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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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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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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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The Replisome03:01

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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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Cohesin protein complexes are a molecular glue that holds two sister chromatids together. They play an important role both in mitosis and meiosis. In mitosis, all cohesin complexes present on the chromosomes are removed before the start of the anaphase stage.
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Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level
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Subunit Communication within Dimeric SF1 DNA Helicases.

Binh Nguyen1, John Hsieh2, Christopher J Fischer3

  • 1Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA.

Journal of Molecular Biology
|April 22, 2024
PubMed
Summary
This summary is machine-generated.

Superfamily 1 helicases require dimerization for activity. Mutations in a key DNA-binding tryptophan abolish helicase function, but heterodimers restore activity, revealing essential subunit interactions.

Keywords:
Helicase activationRepUvrDgain of functionsingle molecule fluorescence

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • Superfamily 1 (SF1) helicases, like E. coli Rep and UvrD, are essential enzymes that unwind DNA.
  • Monomeric SF1 helicases translocate along single-stranded DNA (ssDNA) but require dimerization for full helicase activity.
  • A conserved tryptophan residue in the ssDNA binding site is crucial for helicase function.

Purpose of the Study:

  • To investigate the role of a conserved tryptophan residue in SF1 helicase activity.
  • To elucidate the mechanism of helicase activation through dimerization.
  • To understand subunit communication and specificity in Rep and UvrD heterodimers.

Main Methods:

  • Site-directed mutagenesis to create tryptophan-to-alanine mutants (Rep(W250A), UvrD(W256A)).
  • Ensemble and single-molecule biochemical assays to measure DNA translocation and helicase activity.
  • Fluorescence spectroscopy to monitor subunit interactions within dimers.

Main Results:

  • Mutation of the conserved tryptophan abolished helicase activity in both Rep and UvrD monomers.
  • Mutant monomers retained ssDNA translocase activity but with reduced rates and processivity.
  • Helicase activity was restored in Rep(W250A)/wtRep and UvrD(W256A)/wtUvrD heterodimers.
  • ATPase activity in both subunits of the dimer is necessary for helicase function.
  • Specific interactions between subunits are required, as Rep(W250A) could not activate wtUvrD, and vice versa.
  • Trp fluorescence signals indicated communication within the Rep dimer.

Conclusions:

  • The conserved tryptophan is essential for helicase activity, likely by facilitating base stacking and/or proper dimer formation.
  • Helicase activity is achieved through specific heterodimer formation, requiring contributions from both subunits.
  • These findings support subunit-switching mechanisms for dimeric helicase function and highlight the importance of inter-subunit communication.