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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...
The DNA Replication Fork01:02

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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...
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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...
S-Cdk Initiates DNA Replication02:38

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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 replication.
S-Cdk Initiates DNA Replication02:38

S-Cdk Initiates DNA Replication

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 replication.

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DNA methyltransferase 1 knockdown activates a replication stress checkpoint.

Alexander Unterberger1, Stephen D Andrews, Ian C G Weaver

  • 1Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec, Canada H3G 1Y6.

Molecular and Cellular Biology
|October 4, 2006
PubMed
Summary

Depleting DNA methyltransferase 1 (DNMT1) triggers a DNA damage response via ATR, halting replication. This occurs when DNMT1 is removed from replication forks, not just inhibited.

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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
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Area of Science:

  • Epigenetics
  • Molecular Biology
  • Cellular Biology

Background:

  • DNA methyltransferase 1 (DNMT1) is crucial for maintaining DNA methylation patterns during cell division.
  • The coordination of DNA methylation and replication is vital for epigenetic programming.
  • Previous studies noted DNMT1 depletion inhibits DNA replication, but the underlying mechanism was unclear.

Purpose of the Study:

  • To elucidate the mechanism by which DNMT1 depletion inhibits DNA replication.
  • To investigate the role of genotoxic stress checkpoint proteins in response to DNMT1 loss.

Main Methods:

  • Depletion of DNMT1 using antisense or small interfering RNA (siRNA).
  • Assessment of checkpoint kinase (Chk1/Chk2) phosphorylation and gammaH2AX focus formation.
  • Analysis of cell division control protein 25a (CDC25a) degradation.
  • Utilizing ataxia telangiectasia mutated-Rad3-related (ATR) knockdown and ATR-deficient Seckel syndrome patient cells.
  • Expression of wild-type and catalytically inactive DNMT1.
  • Treatment with 5-aza-deoxycytidine (5-aza-CdR).

Main Results:

  • DNMT1 depletion activates ATR-dependent genotoxic stress responses, including Chk1/Chk2 phosphorylation, gammaH2AX formation, and CDC25a degradation.
  • ATR knockdown or deficiency prevents the replication inhibition and global hypomethylation caused by DNMT1 depletion.
  • The response is independent of DNMT1's catalytic activity but sensitive to DNMT1 depletion itself.
  • Short-term 5-aza-CdR treatment does not elicit this response, suggesting removal from replication forks is key.

Conclusions:

  • The removal of DNMT1 from replication forks, rather than catalytic inhibition, triggers a DNA damage response mediated by ATR.
  • This mechanism highlights a critical link between DNA replication, epigenetic maintenance, and the DNA damage response pathway.
  • Understanding this pathway is essential for comprehending how disruptions in DNA methylation maintenance impact genome stability.