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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.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
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ADP/ATP carrier or AAC protein is the most abundant carrier protein in the inner mitochondrial membrane. It transports large quantities of ADP and ATP, equivalent to the average human body weight, every day. Among other transporters, ACC protein is one of the best-studied members of the mitochondrial carrier protein family. The ADP/ATP carrier protein comprises two transmembrane helices connected to a loop and a single alpha-helix on the matrix side. It switches between two conformational...
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Vesicles incorporate different coat protein subunits in different cell locations, which changes the properties of the coat, such as the shape and geometry of the transport vesicles. Thus, vesicle coat proteins also play a significant role in cargo selection.
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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Related Experiment Video

Updated: Jan 11, 2026

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids
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PCNA is a nucleotide exchange factor for the clamp loader ATPase complex.

Joshua Pajak1, Jacob T Landeck1, Xingchen Liu1

  • 1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605.

Proceedings of the National Academy of Sciences of the United States of America
|November 13, 2025
PubMed
Summary

The Saccharomyces cerevisiae clamp loader, Replication Factor C (RFC), tightly binds ADP. The sliding clamp, Proliferating Cell Nuclear Antigen (PCNA), acts as a nucleotide exchange factor, facilitating ADP release for RFC recycling.

Keywords:
AAA+ ATPaseclamp loadercryo-EM

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • All life depends on loading ring-shaped sliding clamp protein complexes onto DNA.
  • Sliding clamp loaders, like Replication Factor C (RFC), are conserved AAA+ ATPases essential for this process.
  • The mechanism of clamp loader recycling after multiple loading events remains poorly understood.

Purpose of the Study:

  • To investigate the recycling mechanism of the Saccharomyces cerevisiae clamp loader, RFC.
  • To elucidate the role of the sliding clamp, Proliferating Cell Nuclear Antigen (PCNA), in RFC function.
  • To understand how RFC releases ADP to prepare for subsequent clamp loading cycles.

Main Methods:

  • X-ray crystallography to determine the structures of RFC in the absence of PCNA or nucleotide.
  • Molecular dynamics simulations to analyze RFC-PCNA interactions and nucleotide exchange.
  • Biochemical assays to validate the functional implications of structural findings.

Main Results:

  • RFC structures reveal tight ADP binding in multiple ATPase active sites, indicating regulated nucleotide exchange.
  • PCNA binding induces rapid ADP exchange, suggesting PCNA acts as a nucleotide exchange factor (NEF).
  • PCNA likely pries apart RFC subunits, facilitating ADP release and enabling RFC recycling.

Conclusions:

  • PCNA functions as a NEF for RFC, promoting efficient ADP release and clamp loader recycling.
  • This substrate-mediated NEF mechanism prevents premature DNA binding by RFC before PCNA loading.
  • The findings provide critical insights into the regulation and efficiency of DNA replication machinery.