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Absolute quantification of DNA damage response proteins.

Shun Matsuda1, Tsuyoshi Ikura2, Tomonari Matsuda3,4

  • 1Research Center for Environmental Quality Management, Kyoto University, 1-2, Yumihama, Otsu, Shiga, 5200811, Japan.

Genes and Environment : the Official Journal of the Japanese Environmental Mutagen Society
|December 19, 2023
PubMed
Summary

This study quantifies DNA damage response (DDR) proteins, revealing higher levels of non-homologous end joining proteins than homologous recombination proteins. It also shows increased MDC1 chromatin affinity after DNA damage.

Keywords:
Absolute quantificationChromatin affinityDNA damage responseLC-MS/MSMDC1γH2AX

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA damage response (DDR) and repair are crucial for maintaining genetic integrity.
  • Sensor proteins initiate DDR by recognizing DNA double-strand breaks (DSBs).
  • Traditional research focused on individual protein functions, lacking quantitative analysis.

Purpose of the Study:

  • To absolutely quantify DNA damage response and repair proteins.
  • To analyze changes in protein chromatin affinity after DNA damage.
  • To provide quantitative insights into protein dynamics during DDR.

Main Methods:

  • Absolute quantification of DDR and repair proteins in EPC2-hTERT cells.
  • Biochemical fractionation to assess protein chromatin affinity.
  • Analysis of MDC1 and γH2AX dynamics post-DNA damage induction with neocarzinostatin (NCS).

Main Results:

  • H2AX showed the highest intracellular abundance (1.93 × 10^6 molecules/cell).
  • Non-homologous end joining (NHEJ) proteins were significantly more abundant than homologous recombination (HR) proteins.
  • MDC1 demonstrated increased chromatin affinity after DNA damage, peaking at 1 hour post-NCS exposure.

Conclusions:

  • The study offers quantitative insights into protein dynamics within the DNA damage response.
  • Findings highlight differential protein abundance and chromatin binding dynamics.
  • The developed approach enables quantitative analysis of DDR protein behavior.