Related Experiment Video

Updated: Aug 18, 2026

Detection of Protein Ubiquitination
09:00

Detection of Protein Ubiquitination

Published on: August 19, 2009

DDB2, the xeroderma pigmentosum group E gene product, is directly ubiquitylated by Cullin 4A-based ubiquitin ligase

Noriyuki Matsuda1, Keiko Azuma, Masafumi Saijo

  • 1Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

DNA Repair
|April 7, 2005
PubMed

Insights

Xeroderma pigmentosum (XP) is a DNA repair disorder. This study shows the Cul4A-DDB complex directly ubiquitylates DDB2, a protein implicated in XP-E, clarifying a key step in DNA damage response.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Xeroderma pigmentosum (XP) is a genetic disorder causing UV hypersensitivity and skin cancer due to DNA repair defects.
  • Mutations in damaged-DNA binding protein 2 (DDB2) cause XP complementation group E (XP-E).
  • The DDB complex (DDB1-DDB2) binds damaged DNA and is crucial for nucleotide excision repair.

Purpose of the Study:

  • To investigate the direct interaction and ubiquitylation of DDB2 by the Cul4A E3 ubiquitin ligase complex.
  • To determine if the Cul4A complex directly ubiquitylates DDB2 in vitro.

Main Methods:

  • Screening for proteins interacting with Cullin 4A (Cul4A).
  • Immunoprecipitation to confirm protein interactions.
  • Purification of the Cul4A-containing E3 complex.
  • In vitro ubiquitylation assays using purified components.

Main Results:

  • Identified DDB1 and DDB2 as interacting partners of Cul4A.
  • Confirmed Cul4A interacts with both DDB1 and DDB2.
  • Reconstituted DDB2 ubiquitylation in vitro using the purified Cul4A-containing E3 complex, demonstrating direct ubiquitylation.
  • Showed that the XP-E associated K244E mutation in DDB2 does not affect its ubiquitylation.

Conclusions:

  • The DDB-Cul4A E3 complex directly ubiquitylates DDB2.
  • This finding clarifies a critical step in the DNA damage response pathway relevant to XP-E.
  • The K244E mutation, linked to XP-E, does not impair DDB2 ubiquitylation by this complex.

More Related Videos

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Related Concept Videos

Nucleotide Excision Repair01:38

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview
Abnormal Proliferation02:23

Abnormal Proliferation

Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the daughter...
Regulated Protein Degradation02:58

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
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...
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.