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Updated: Jul 31, 2026
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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Purification and characterization of human DNA damage checkpoint Rad complexes
L A Lindsey-Boltz1, V P Bermudez, J Hurwitz
1Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Summary
Human checkpoint Rad proteins, including hRad17-RFC and the 9-1-1 complex, act as DNA damage sensors. These proteins are crucial for arresting cell cycle progression, ensuring genetic stability during DNA repair.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Damage Response
Background:
- Checkpoint Rad proteins initiate the DNA damage response, arresting cell cycle progression.
- This ensures accurate transmission of genetic material to daughter cells.
- Understanding the damage sensor function of human checkpoint Rad proteins is critical.
Purpose of the Study:
- To biochemically characterize the damage sensor function of human checkpoint Rad proteins.
- To investigate the interactions between hRad17-RFC and the checkpoint 9-1-1 complex.
Main Methods:
- Purification of a heteropentameric hRad17-RFC complex.
- Purification of a heterotrimeric hRad9-hHus1-hRad1 (9-1-1) complex.
- Biochemical assays to assess DNA binding and ATPase activity of hRad17-RFC.
Main Results:
- hRad17-RFC binds preferentially to primed DNA.
- hRad17-RFC exhibits ATPase activity, stimulated by primed and single-stranded DNA.
- hRad17-RFC forms a complex with the 9-1-1 heterotrimer, similar to replication machinery components.
Conclusions:
- These findings provide biochemical evidence for the role of checkpoint Rad proteins as DNA damage sensors.
- The study supports models of checkpoint Rads in the human DNA damage checkpoint pathway.
- The interaction between hRad17-RFC and the 9-1-1 complex is key to checkpoint signaling.
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