Dataset Information

Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

ABSTRACT: The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cancerous cells. Human RAD51 is, therefore, an important and highly coveted anticancer drug target. Biochemical, biophysical, and structural studies of the human RAD51 and establishment of its structure-activity relationship require purification of large quantities of protein. In this chapter we describe a robust method for expression and purification of human RAD51 and the methods for assessing its activity based on the single-strand DNA-binding stoichiometry and its capacity to carry out the DNA strand exchange reaction.

SUBMITTER: Subramanyam S

PROVIDER: S-EPMC6033273 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

Subramanyam Shyamal S Spies Maria M

Methods in enzymology 20180109

The RAD51 DNA strand exchange protein plays an important role in maintaining the integrity of the human genome. It promotes homology-directed DNA repair by exchanging strands between the damaged and the intact DNA molecules. It also plays an important role in stabilizing distressed DNA replication forks. When overexpressed or misregulated, however, RAD51 contributes to "rogue," genome destabilizing events that can lead to cancer, cell death, and to acquisition of chemotherapy resistance by cance ...[more]

PMID: 29458757

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Project description:Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor alpha mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 microM Zn(2+). The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex.

Dataset Information

Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

Publications

Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein.

Similar Datasets

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