Related Experiment Video
Updated: Dec 7, 2025
12:26
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
19.2K
CK2 kinase-mediated PHF8 phosphorylation controls TopBP1 stability to regulate DNA replication.
Haihua Feng1, Jingchen Lu1,2, Xiaotian Song1
1Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USA.
Nucleic Acids Research
|October 3, 2020
Summary
The histone demethylase PHF8 binds to the ATR activator TopBP1, stabilizing it and preventing degradation. This interaction is crucial for maintaining DNA replication and restarting replication forks after damage.
Area of Science:
- Cellular Biology
- Molecular Biology
- Genetics
Background:
- ATR is a key regulator of the DNA-damage response.
- ATR activation depends on its activator, topoisomerase IIβ-binding protein 1 (TopBP1).
- The regulation of TopBP1 and its impact on DNA replication are not fully understood.
Purpose of the Study:
- To investigate the interaction between TopBP1 and the histone demethylase PHF8.
- To elucidate the mechanism by which PHF8 regulates TopBP1.
- To understand the role of the TopBP1/PHF8 interaction in DNA replication.
Main Methods:
- Co-immunoprecipitation to identify protein interactions.
- Site-directed mutagenesis to study phosphorylation effects.
- Western blotting to assess protein levels and ubiquitination.
- Cell cycle analysis.
- Replication fork stability assays.
Main Results:
- A specific interaction between TopBP1 and PHF8 was identified, mediated by TopBP1's BRCT 7+8 domain and PHF8 phosphorylation at Ser854.
- This interaction is cell-cycle regulated and dependent on CK2-mediated phosphorylation of PHF8.
- PHF8 prevents TopBP1 ubiquitination and degradation by the E3 ligase UBR5, thereby regulating TopBP1 protein levels.
- PHF8 phosphorylation at Ser854 (PHF8pS854) contributes to TopBP1 stability and the DNA replication checkpoint.
- Both TopBP1 and PHF8 are essential for efficient replication fork restart.
Conclusions:
- PHF8 acts as a TopBP1-binding protein.
- PHF8 stabilizes TopBP1 by inhibiting its degradation, which is critical for DNA replication.
- The PHF8-TopBP1 interaction plays a significant role in maintaining DNA replication fidelity and checkpoint control.
More Related Videos
Related Concept Videos
02:37DNA Damage can Stall the Cell Cycle
9.8K
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...
9.8K
02:37Restarting Stalled Replication Forks
6.1K
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,...
6.1K
02:38S-Cdk Initiates DNA Replication
5.2K
The cell cycle is a series of events leading to DNA duplication followed by the division of cell content to form two daughter cells. The cell cycle progresses in four stages—the cell increases in size (gap 1 or G1-phase), duplicates its DNA (synthesis or S-phase), prepares to divide (gap 2 or G2-phase), and divides (mitosis or M-phase).
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
5.2K
01:23Negative Regulator Molecules
37.7K
Positive regulators allow a cell to advance through cell cycle checkpoints. Negative regulators have an equally important role as they terminate a cell’s progression through the cell cycle—or pause it—until the cell meets specific criteria.
37.7K
02:02DNA Topoisomerases
34.0K
Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
Types and Mechanism of action
Topoisomerases are divided into two main types. ...
Types and Mechanism of action
Topoisomerases are divided into two main types. ...
34.0K
02:34Inhibition of Cdk Activity
5.4K
The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex...
5.4K