Related Experiment Video

Updated: Jul 3, 2026

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites
09:39

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

Published on: August 2, 2024

Identification of phosphorylation sites on transcription factor Sp1 in response to DNA damage and its accumulation at

Satoko Iwahori1, Yoshihiro Yasui, Ayumi Kudoh

  • 1Division of Virology, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan.

Cellular Signalling
|July 16, 2008
PubMed

Insights

DNA damage triggers Sp1 protein phosphorylation, particularly at Ser-101, involving ATM and ATR kinases. This phosphorylation appears linked to DNA repair, not transcriptional regulation.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Genetics

Background:

  • DNA damage is a critical cellular event impacting genomic stability.
  • The Sp1 transcription factor plays a role in gene expression.
  • Post-translational modifications, like phosphorylation, regulate protein function.

Purpose of the Study:

  • To investigate the role of Sp1 phosphorylation in response to DNA damage.
  • To identify the specific residues phosphorylated on Sp1 and the kinases involved.
  • To determine the functional consequence of Sp1 phosphorylation in DNA repair and transcriptional activity.

Main Methods:

  • Treatment of cells with ionizing radiation (IR), UV irradiation, and hydroxyurea (HU).
  • Western blotting to detect Sp1 phosphorylation at specific residues (Ser-56, Ser-101).
  • RNA interference (siRNA) to silence Sp1, ATM, and ATR expression.
  • Confocal laser microscopy to visualize protein co-localization.

Main Results:

  • Ionizing radiation-induced DNA double-strand breaks (DSBs) cause ATM-dependent phosphorylation of Sp1 at Ser-56 and Ser-101.
  • Replicative stress (UV or HU) induces ATM/ATR-dependent phosphorylation of Sp1 solely at Ser-101.
  • Sp1 phosphorylation at Ser-101 did not alter its transcriptional activity.
  • Phosphorylated Sp1 (pSp1) co-localizes with phosphorylated ATM at DNA damage sites.

Conclusions:

  • Sp1 phosphorylation, particularly at Ser-101, is a general DNA damage response mediated by ATM and ATR.
  • Phosphorylated Sp1 is recruited to DNA damage sites, suggesting a role in DNA repair.
  • Sp1 phosphorylation's primary role in DNA damage response may be in DNA repair rather than transcriptional regulation.

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

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

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...
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...
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
Long-patch Base Excision Repair01:02

Long-patch Base Excision Repair

Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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.
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...