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Related Concept Videos

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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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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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 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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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...
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Related Experiment Video

Updated: Aug 4, 2025

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
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"Helicase" Activity promoted through dynamic interactions between a ssDNA translocase and a diffusing SSB protein.

Kacey N Mersch1, Joshua E Sokoloski1,2, Binh Nguyen1

  • 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110-1093.

Proceedings of the National Academy of Sciences of the United States of America
|April 3, 2023
PubMed
Summary

The Pif1 translocase protein pushes human replication protein A (hRPA) along single-stranded DNA, disrupting duplex DNA. This reveals a mechanism for directional DNA unwinding by combining a translocase and an SSB protein.

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DNA motorsRPAdynamicsoptical tweezerssingle-molecule fluorescence

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Replication protein A (RPA) is a crucial eukaryotic single-stranded DNA-binding protein involved in genome maintenance.
  • RPA exhibits high-affinity binding to ssDNA and can diffuse along it, potentially disrupting short duplex DNA regions.
  • Understanding RPA's dynamic interactions is key to comprehending DNA repair and replication processes.

Purpose of the Study:

  • To investigate the mechanism by which the *S. cerevisiae* Pif1 protein interacts with and moves human RPA (hRPA) on single-stranded DNA (ssDNA).
  • To determine if Pif1 can utilize its translocation activity to displace hRPA into duplex DNA and induce DNA unwinding.
  • To elucidate the combined roles of ssDNA translocases and SSB proteins in DNA unwinding and genome maintenance.

Main Methods:

  • Single-molecule total internal reflection fluorescence microscopy.
  • Optical trapping combined with fluorescence approaches.
  • Biochemical assays to measure Pif1 translocation and hRPA displacement.

Main Results:

  • *S. cerevisiae* Pif1 uses ATP-dependent 5' to 3' translocation to push hRPA along ssDNA at significant rates.
  • Pif1 can displace hRPA from ssDNA into duplex DNA, causing stable disruption of at least 9 base pairs.
  • Demonstrated that Pif1's translocation activity can drive hRPA into duplex DNA, leading to unwinding.

Conclusions:

  • The dynamic nature of hRPA allows for reorganization even when tightly bound to ssDNA.
  • A mechanism for directional DNA unwinding is presented, involving a ssDNA translocase pushing an SSB protein.
  • Processive DNA helicase function requires transient DNA melting (by hRPA) and directional ssDNA translocation (by Pif1), which can be uncoupled using separate proteins.