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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
Topological Features of Histone H2A Monoubiquitination
A A Kudriaeva1, V M Lipkin2, A A Belogurov2,3
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. anna.kudriaeva@gmail.com.
Abstract:
The cellular response to DNA damage protects the essential information stored in the genome. This mechanism is crucial in terms of the cancer prevention and aging progression. The DNA damage response (DDR) consists of a complex network controlling the cell cycle and multiple mechanisms of the DNA repair. The DDR disruption is a cornerstone feature of the tumor cells, which allows them to enhance beneficial mutations that prevent successful disease treatment. The important checkpoints of the DDR are currently poorly understood due to the complexity and diversity of the DNA repair machinery. Histone ubiquitination is intensively involved in the repair of the double-stranded DNA breaks. This post-translational modification is known to be a key factor in the recruitment of the repair factors to the DNA damage sites. Here, the crucial role of the ubiquitin lysine residue K27 in the process of histone H2A monoubiquitination mediated by the ubiquitin ligase RNF168 has been showed. The presented data suggest forced and intensive diffusion of ubiquitin from the cytoplasm to the nucleus, which is characterized by the dynamic equilibrium less than 10 min. The comparison of the turnover rate of the wild-type ubiquitin and its variant with a single functional lysine residue K27 suggests an important role of the ubiquitin deposition as a covalent conjugate with histone H2A in terms of the stability of the entire ubiquitinome.
Insights
DNA damage response (DDR) is vital for preventing cancer and aging. This study highlights histone ubiquitination, specifically ubiquitin lysine K27, as crucial for DNA repair and genome stability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The DNA damage response (DDR) is essential for maintaining genomic integrity, preventing cancer, and regulating aging.
- Disruptions in DDR pathways are characteristic of cancer cells, enabling uncontrolled proliferation and treatment resistance.
- Histone ubiquitination plays a critical role in recruiting DNA repair factors to damage sites, particularly in double-stranded DNA break repair.
Purpose of the Study:
- To elucidate the specific role of ubiquitin lysine K27 in histone H2A monoubiquitination during DNA repair.
- To investigate the dynamics of ubiquitin diffusion between the cytoplasm and nucleus in response to DNA damage.
- To assess the contribution of ubiquitin deposition as a histone H2A conjugate to the stability of the ubiquitinome.
Main Methods:
- Utilized ubiquitin ligase RNF168 to mediate histone H2A monoubiquitination.
- Compared the turnover rates of wild-type ubiquitin and a K27-mutant variant.
- Monitored ubiquitin diffusion dynamics between cellular compartments.
Main Results:
- Demonstrated the critical involvement of ubiquitin lysine K27 in RNF168-mediated histone H2A monoubiquitination.
- Observed rapid ubiquitin diffusion (dynamic equilibrium <10 min) from cytoplasm to nucleus.
- Identified ubiquitin deposition onto histone H2A as a key factor for ubiquitinome stability.
Conclusions:
- Ubiquitin lysine K27 is essential for efficient histone H2A monoubiquitination in DNA repair.
- The dynamic ubiquitin exchange between cytoplasm and nucleus is rapid and crucial.
- Ubiquitin conjugation to histone H2A significantly contributes to the overall stability of the cellular ubiquitinome.
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