Related Experiment Video

Updated: Apr 14, 2026

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
12:43

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Published on: November 12, 2017

11.6K

A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage

X Chen1,2, L Bosques1,2, P Sung3

  • 1Department of Pediatrics, Section of Hematology/Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.

Oncogene
|April 21, 2015
PubMed

Insights

Fanconi anemia (FA) cells resist hydroxyurea (HU) unless FANCD2 is absent. FANCD2 and RAD51 promote DNA repair via PCNA monoubiquitination and translesion synthesis, independent of FA pathway ubiquitination.

Area of Science:

  • Genetics
  • Molecular Biology
  • DNA Repair

Background:

  • Fanconi anemia (FA) is a genetic disorder causing bone marrow failure and cancer susceptibility.
  • The FA pathway, centered on FANCD2, is crucial for DNA damage response, particularly to crosslinking agents.
  • FA cells typically show sensitivity to DNA crosslinkers but differential responses to replication blockers like hydroxyurea (HU).

Purpose of the Study:

  • To investigate the differential response of FA cells to hydroxyurea (HU) compared to mitomycin C (MMC).
  • To elucidate the role of FANCD2 and RAD51 in DNA repair mechanisms, specifically PCNA monoubiquitination and translesion synthesis (TLS), in response to HU.
  • To determine if FANCD2 ubiquitination and homologous recombination (HR) are required for these repair processes.

Main Methods:

  • Cell culture and treatment with HU and MMC.
  • Western blotting to detect protein ubiquitination (FANCD2, PCNA).
  • Immunoprecipitation to analyze protein complex formation (FANCD2, RAD51, RAD18).
  • siRNA knockdown of FANCD2, RAD51, and PolH.
  • Assessment of PolH chromatin localization and cell proliferation assays.

Main Results:

  • FA cells lacking FANCD2 expression are sensitive to HU, unlike other FA cells.
  • FANCD2 and RAD51 form a complex upon HU exposure, independent of FANCD2 monoubiquitination.
  • Non-ubiquitinated FANCD2 supports PCNA monoubiquitination, and RAD51 is required for this process, independent of BRCA2 and HR.
  • PolH chromatin localization is reduced in FANCD2 or RAD51 deficient cells, and PolH knockdown causes HU sensitivity.
  • FANCD2 and RAD51 play a role in PCNA monoubiquitination and TLS in an HR-independent manner during HU treatment.

Conclusions:

  • FANCD2 and RAD51 mediate PCNA monoubiquitination and translesion synthesis in response to HU through a mechanism independent of FANCD2 ubiquitination and homologous recombination.
  • This pathway represents a non-canonical function of the FA pathway, distinct from its response to mitomycin C.
  • These findings highlight a novel role for FANCD2 and RAD51 in managing replication stress induced by agents like HU.

More Related Videos

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage10:59

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Published on: August 21, 2021

4.2K
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins10:24

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Published on: September 28, 2012

14.7K

Related Concept Videos

DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

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...
10.4K
DNA Damage Can Stall the Cell Cycle02:36

DNA Damage Can Stall the Cell Cycle

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...
3.4K
Overview of DNA Repair02:25

Overview of DNA Repair

In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...
35.5K
Overview of DNA Repair02:25

Overview of DNA Repair

10.6K
Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview
42.8K
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...
5.9K