Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Protein Complex Assembly02:41

Protein Complex Assembly

Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Protein Complex Assembly02:41

Protein Complex Assembly

Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
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...
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...
Immunoprecipitation01:20

Immunoprecipitation

Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Structure of the pre-initiation complex explains CMGE biogenesis.

Nature·2026
Same author

Targeting Cancer-Specific Mutations with RNA-Triggered Chromatin Shredding.

Nature·2026
Same author

Selective Elimination of TP53 Mutant Cells by Transcript-Activated Chromatin Shredding.

bioRxiv : the preprint server for biology·2026
Same author

Biochemical reconstitution of sister chromatid cohesion establishment during DNA replication.

Molecular cell·2025
Same author

Replisome passage through the cohesin ring.

Cell·2025
Same author

S-phase checkpoint protects from aberrant replication fork processing and degradation.

Nucleic acids research·2025
Same journal

An accessible, absorbance-based plate reader assay to assess cumulative exposure of blood plasma & serum to thawed conditions.

Methods (San Diego, Calif.)·2026
Same journal

EC-isHCR: A rapid method for in situ hybridization chain reaction in diverse animal samples.

Methods (San Diego, Calif.)·2026
Same journal

Single-Molecule methods to investigate mechanisms of transcription by RNA polymerase of Mycobacterium tuberculosis.

Methods (San Diego, Calif.)·2026
Same journal

Detection and sequencing of Usutu virus during mosquito surveillance: Use of multiple assays and techniques for identification at low levels.

Methods (San Diego, Calif.)·2026
Same journal

Experimental validation of an AI-driven digital healthcare platform for oral health behavior and plaque assessment among vietnamese children.

Methods (San Diego, Calif.)·2026
Same journal

Zeta potential: An efficient and cost-effective alternative for investigating cell-surface interactions.

Methods (San Diego, Calif.)·2026
See all related articles

Related Experiment Video

Updated: May 21, 2026

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
07:27

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Published on: April 29, 2010

Pre-replicative complex assembly with purified proteins.

Amina Mehanna1, John F X Diffley

  • 1Clare Hall Laboratories, Cancer Research UK, South Mimms, UK.

Methods (San Diego, Calif.)
|June 27, 2012
PubMed
Summary
This summary is machine-generated.

This study details the in vitro reconstitution of pre-replicative complex (pre-RC) formation, crucial for eukaryotic DNA replication. It explains how minichromosome maintenance proteins (Mcm2-7) are loaded, ensuring DNA replication occurs only once per cell cycle.

More Related Videos

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
08:12

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Published on: January 3, 2019

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach
09:57

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Published on: December 17, 2016

Related Experiment Videos

Last Updated: May 21, 2026

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
07:27

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Published on: April 29, 2010

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
08:12

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

Published on: January 3, 2019

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach
09:57

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Published on: December 17, 2016

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Biochemistry

Background:

  • Eukaryotic DNA replication initiation requires the assembly of pre-replicative complexes (pre-RCs).
  • Pre-RC formation is tightly regulated and restricted to the G1 phase to ensure genomic stability.
  • The minichromosome maintenance (Mcm2-7) complex is a core component loaded during pre-RC assembly.

Purpose of the Study:

  • To describe a detailed in vitro method for reconstituting pre-RC formation.
  • To investigate the biochemical mechanisms underlying Mcm2-7 loading onto DNA.
  • To provide a foundation for studying subsequent DNA replication steps.

Main Methods:

  • Purification of key pre-RC proteins: origin recognition complex (ORC), Cdc6, Cdt1, and Mcm2-7.
  • In vitro biochemical assays to reconstitute pre-RC assembly in the presence of ATP.
  • Analysis of Mcm2-7 loading mechanism and stoichiometry.

Main Results:

  • Successfully reconstituted the ATP-dependent loading of Mcm2-7 onto DNA in vitro.
  • Demonstrated that Mcm2-7 is loaded as a symmetrical head-to-head double hexamer.
  • Established a reliable method for studying the dynamics of pre-RC formation.

Conclusions:

  • The described in vitro system accurately recapitulates Mcm2-7 loading during pre-RC formation.
  • This methodology facilitates detailed biochemical analysis of DNA replication initiation.
  • The findings contribute to understanding cell cycle control and preventing replication errors.