Related Experiment Video

Updated: Jun 5, 2026

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
10:50

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Published on: March 14, 2019

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3

Maarten Hoek1, Michael P Myers, Bruce Stillman

  • 1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

The Journal of Biological Chemistry
|January 7, 2011
PubMed

Insights

Chromatin assembly factor CAF-1 is vital for DNA replication and repair. Its N-terminal region is not essential for these functions, despite affecting proliferating cell nuclear antigen binding.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Epigenetics

Background:

  • Chromatin assembly factor CAF-1 is crucial for histone deposition during DNA replication and repair in human cells.
  • CAF-1 depletion leads to replication fork arrest, cell cycle checkpoint activation, and chromatin structure defects.
  • CAF-1 also plays a role in maintaining gene expression states and chromatin assembly post-DNA repair.

Purpose of the Study:

  • To investigate the functional significance of the N-terminal region of CAF-1 in human cells.
  • To identify CAF-1 interacting proteins involved in DNA replication and repair.
  • To elucidate the role of specific CAF-1 domains in chromatin assembly processes.

Main Methods:

  • Generation of RNAi-resistant CAF-1 variants.
  • Analysis of proliferating cell nuclear antigen (PCNA) binding.
  • Tandem affinity purification to identify CAF-1 interacting proteins.
  • In vitro kinase assays and interaction studies.

Main Results:

  • The N-terminal 296 amino acids of CAF-1 are dispensable for its essential functions in vivo.
  • Truncated CAF-1 showed reduced PCNA binding but maintained chromatin recruitment after DNA damage.
  • CAF-1 directly interacts with KU70/80 and 14-3-3 ζ, and is phosphorylated by DNA-dependent protein kinase.

Conclusions:

  • The N terminus of CAF-1 is not essential for DNA replication- and repair-coupled chromatin assembly.
  • CAF-1 interacts with key DNA repair factors, highlighting its integration into DNA damage response pathways.
  • Specific domains of CAF-1 mediate interactions with PCNA and the DNA repair machinery.

More Related Videos

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay10:05

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Published on: January 16, 2017

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes10:05

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Published on: August 13, 2012

Related Concept Videos

Assembly of Signaling Complexes01:30

Assembly of Signaling Complexes

Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
Catenins01:23

Catenins

Catenins are characterized by multiple binding domains and dynamic structures that allow them to function as linker proteins in cell junction complexes. All catenins, except α-catenin, contain a characteristic protein sequence called the armadillo repeat and are therefore also called armadillo proteins.
Catenins in Cell Junctions
Catenins bind to cell adhesion molecules such as cadherins and link them to different cytoskeletal proteins depending on the type of cell junction. At the adherens...
Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Protein Networks02:26

Protein Networks

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
cAMP-dependent Protein Kinase Pathways01:25

cAMP-dependent Protein Kinase Pathways

Cyclic Adenosine Monophosphate (cAMP) is an essential second messenger that activates protein kinase A (PKA) and regulates various biological processes. A single epinephrine molecule binds to GPCR and activates several heterotrimeric G proteins, each stimulating multiple adenylyl cyclase, amplifying the signal, and synthesizing large numbers of cAMP molecules. Small changes in cAMP concentration affect PKA activity. The binding of four cAMP molecules induces a conformational change in PKA,...