Centrosomal localization of DNA damage checkpoint proteins
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Updated: Jul 18, 2026
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Published on: September 28, 2012
Suisheng Zhang1, Peter Hemmerich, Frank Grosse
1Leibniz Institute for Age Research, Jena, Germany.
Abstract:
During mitosis, the phosphatidylinositol-3 (PI-3) family-related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA-PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA-PKcs, the catalytic subunit of DNA-PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co-immunoprecipitated with Ku70/86, the DNA binding subunits of DNA-PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of alpha-tubulin foci in the nucleus. Interestingly, gamma-tubulin was also present in the nucleus and co-immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of gamma-tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of gamma-tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response.
Insights
DNA damage checkpoint proteins like ATM and ATR are found on human cell centrosomes. These proteins may regulate microtubule formation and DNA damage response during mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The DNA damage response (DDR) is crucial for maintaining genomic stability.
- Centrosomes play key roles in cell division and microtubule organization.
- ATM and ATR are central kinases in the DDR pathway.
Purpose of the Study:
- To investigate the localization and function of DNA damage checkpoint proteins at centrosomes during mitosis.
- To explore the interplay between DNA damage signaling and centrosome-associated proteins.
Main Methods:
- Immunofluorescence microscopy to visualize protein localization.
- Co-immunoprecipitation assays to study protein interactions.
- Western blotting to assess protein phosphorylation.
- Treatment with DNA-PK inhibitor (vanillin) and induction of DNA damage.
Main Results:
- ATM, ATR, ATRIP, Chk1, and Chk2 were localized to centrosomes during mitosis.
- The DNA-PK inhibitor vanillin enhanced ATM levels at centrosomes and altered Chk2 phosphorylation.
- ATM interacted with DNA-PK subunits (Ku70/86), and this interaction was reduced by vanillin.
- Gamma-tubulin was found in the nucleus and centrosomes, interacting with ATR and BRCA1.
- DNA damage reduced checkpoint proteins at centrosomes but increased gamma-tubulin levels.
Conclusions:
- DNA damage checkpoint proteins are dynamically regulated at centrosomes during mitosis.
- These proteins may influence gamma-tubulin dynamics and microtubule formation at centrosomes.
- Centrosome-associated checkpoint proteins link DNA damage signaling to cell division machinery.
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