التفاصيل البيبلوغرافية
| العنوان: |
Preparation of endogenous TopBP1/Dpb11 and effect on central checkpoint kinase Mec1- Ddc2 (human ATR-ATRIP homolog). |
| المؤلفون: |
Wu, Tengwei1 (AUTHOR), Zhang, Zhihui1 (AUTHOR), Wang, Xuejuan1 (AUTHOR) xuejuan@ustc.edu.cn |
| المصدر: |
Biochemical & Biophysical Research Communications. Sep2019, Vol. 517 Issue 2, p291-296. 6p. |
| مصطلحات موضوعية: |
*DNA replication, *DNA damage |
| مستخلص: |
The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5 μM of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1–Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations. • Establishing highly efficient preparation method for endogenous Dpb11 activator. • The high purity and homogeneity enables structural and functional characterization. • The endogenous Dpb11 stimulates Mec1 kinase activity and promotes conformational changes. • Disclosing Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations. [ABSTRACT FROM AUTHOR] |
| قاعدة البيانات: |
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