Related Experiment Video

Updated: Aug 24, 2026

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG
10:11

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Published on: July 26, 2024

MCM4 shares homology to a replication/DNA-binding domain in CTF and is contacted by pRb

Nicole M R Schmitz1, Kurt Leibundgut, Andreas Hirt

  • 1Department of Clinical Research, University of Bern, Switzerland. Nicole.schmitz@dkf3.unibe.ch

Biochemical and Biophysical Research Communications
|April 15, 2004
PubMed

Insights

The retinoblastoma protein (pRb) controls DNA replication initiation. Cyclin D-dependent kinases (CDKs) phosphorylate pRb, releasing it from chromatin and allowing MCM4 to bind, influencing DNA replication and repair processes.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • DNA Replication

Background:

  • The retinoblastoma protein (pRb) is a key regulator of the cell cycle, primarily known for its role in limiting DNA replication.
  • Understanding the precise mechanisms by which pRb controls cell cycle progression and interacts with other cellular machinery is crucial for comprehending normal and aberrant cell growth.

Purpose of the Study:

  • To investigate the role of Cdk4,6-cyclin D in pRb phosphorylation and subsequent release from chromatin.
  • To identify novel binding partners of under-phosphorylated pRb.
  • To elucidate the functional implications of pRb interactions in DNA replication and repair.

Main Methods:

  • Immunoblotting of in vitro kinase assays to assess pRb phosphorylation.
  • Immunoprecipitation of differential cell extractions to analyze pRb chromatin association.
  • Two-hybrid assay using LexA-Rb(561-660) to identify pRb binding proteins.
  • Sequence analysis to identify conserved motifs and predict protein function.
  • Immunodetection of protein complexes in cell extracts.

Main Results:

  • Cdk4,6-cyclin D phosphorylates pRb at Ser-608, a critical step for pRb release from chromatin in Nalm-6 cells.
  • MCM4 was identified as a novel binding partner for under-phosphorylated pRb.
  • A conserved motif in MCM4, homologous to CTF/NF-I domains, suggests a role in DNA binding and replication.
  • A pRb-MCM4-CTF/NF-I complex was detected, supporting a model where CTF/NF-I facilitates MCM4 binding to pRb.
  • The identified motif in MCM4 is also present in XPD, suggesting collaborative roles in DNA repair and transcription.

Conclusions:

  • pRb phosphorylation by Cdk4,6-cyclin D is essential for its dissociation from chromatin, thereby regulating DNA replication initiation.
  • MCM4 is a novel interaction partner of pRb, linking pRb function to the MCM complex and DNA replication machinery.
  • The findings suggest a coordinated mechanism involving pRb, MCM4, and CTF/NF-I in regulating DNA replication and potentially DNA repair pathways.

More Related Videos

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase07:37

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Published on: September 27, 2024

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy11:40

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Published on: June 25, 2013

Related Concept Videos

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...
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

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, a...
The Replisome03:01

The Replisome

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 the...
The Replisome03:01

The Replisome

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 the...
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...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...