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Analyzing DNA replication dynamics of genotoxin-treated cells using velocity sedimentation
Tovah A Day1, Chris Sproul, Marila Cordeiro-Stone
1Dana Farber Cancer Institute, Boston, MA, USA. tovah_day@dfci.harvard.edu
Methods in Molecular Biology (Clifton, N.J.)
|August 27, 2011
Summary
DNA damage can halt cell cycle progression by impacting DNA replication initiation and elongation. A novel velocity sedimentation method distinguishes these effects, aiding S-phase checkpoint studies.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA damage can impede S-phase progression through signaling pathways (S-phase checkpoints) or physical blockage of replication forks.
- Current methods for assessing DNA synthesis inhibition do not differentiate between effects on initiation and elongation.
Purpose of the Study:
- To introduce and validate a velocity sedimentation protocol for distinguishing DNA damage effects on replication initiation and elongation.
- To provide a tool for analyzing S-phase checkpoint function and DNA replication dynamics.
Main Methods:
- Labeling replicating DNA with (3)H-thymidine.
- Analyzing nascent single-stranded DNA (ssDNA) size distribution via alkaline sucrose gradient velocity sedimentation.
- Quantifying small nascent ssDNAs to index initiation and growth rates to index elongation.
Main Results:
- The velocity sedimentation method successfully differentiates between inhibition of DNA replication initiation and elongation.
- This technique provides quantitative data on replication dynamics in response to DNA damage.
Conclusions:
- Velocity sedimentation is a valuable tool for dissecting the impact of DNA damage on DNA replication.
- This method enhances the study of S-phase checkpoints and replication fork progression.
