Related Experiment Video

Updated: Jul 18, 2026

Yeast As a Chassis for Developing Functional Assays to Study Human P53
14:57

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Published on: August 4, 2019

p53-Dependent p21 mRNA elongation is impaired when DNA replication is stalled

Melissa Mattia1, Vanesa Gottifredi, Kristine McKinney

  • 1Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

Molecular and Cellular Biology
|December 13, 2006
PubMed

Insights

Hydroxyurea (HU) impairs p21 gene induction by inhibiting transcription elongation, not initiation, in human cells. This suggests the DNA replication checkpoint affects elongation factors, not promoter complex assembly.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • Gene Expression

Background:

  • p53 is a key tumor suppressor protein that regulates genes involved in cell cycle arrest and apoptosis.
  • p21(WAF1/CIP1) is a critical p53 target gene that inhibits cyclin-dependent kinases, promoting cell cycle arrest.
  • Previous studies showed that blocking DNA replication impairs p53-mediated induction of some target genes, including p21.

Purpose of the Study:

  • To investigate the molecular mechanism by which hydroxyurea (HU) impairs p53-mediated p21 induction.
  • To compare the effects of HU (S phase arrest) and daunorubicin (G2 block) on p21 gene expression and p53 activity.

Main Methods:

  • Cell culture and treatment with hydroxyurea (HU) or daunorubicin.
  • Quantitative reverse transcription-PCR to measure p21 mRNA levels.
  • Chromatin immunoprecipitation (ChIP) assays to assess p53 binding and histone modifications.
  • ChIP assays to evaluate the recruitment of transcription initiation factors and RNA polymerase II (RNA Pol II).
  • Analysis of RNA Pol II occupancy and phosphorylation at different gene regions.

Main Results:

  • HU treatment inhibited p21 mRNA transcription, not mRNA stability.
  • HU did not affect p53 binding or histone acetylation at the p21 promoter.
  • Recruitment of TFIID/TATA-binding protein and RNA Pol II to the promoter was similar for HU and daunorubicin.
  • HU significantly impaired transcription elongation of the p21 gene, indicated by reduced RNA Pol II occupancy downstream of the start site.
  • HU treatment led to decreased levels of phosphorylated RNA Pol II (Ser2P) in the p21 gene's downstream region.

Conclusions:

  • The DNA replication checkpoint, triggered by HU, does not impede the assembly of the p21 promoter initiation complex.
  • The replication checkpoint likely signals to factors that regulate the process of transcriptional elongation for the p21 gene.
  • Understanding these mechanisms is crucial for cancer therapy, as p53 and p21 are key players in tumor suppression.

More Related Videos

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase07:27

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Published on: April 29, 2010

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique07:18

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Published on: October 27, 2011

Related Concept Videos

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...
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...
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...
Negative Regulator Molecules01:23

Negative Regulator Molecules

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.
The DNA Replication Fork01:02

The DNA Replication Fork

An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...