Study of the specific protein-protein interaction of the XRCC4-SIRT2 in vitro
Date Issued
2006
Date
2006
Author(s)
Huang, Yi-Shiang
DOI
zh-TW
Abstract
XRCC4 is an essential protein in the mammalian DNA repair system. Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination. In the DNA double strand break (DSB) repair pathway, XRCC4-LigaseIV complex execute the final DNA end-joining reaction. XRCC4 has been known greatly stabilizing LigaseIV, and can stimulate the ligase activity of the DNA LigaseIV.
SIRT2 is a member of the yeast silent information regulator 2 (Sir2) enzyme families. Yeast Sir2 functions as an NAD+-dependent protein deacetylase, and conducts biological functions such as genomic silencing, DNA repair, life span in calorie restriction. Human SIRT2 is a member of yeast Sir2 family. Mammalian Sir2 ortholog is called Sirtuin and classified as NAD+-dependent Histone deacetylase class III. The enzyme activity and the catalytic mechanism of yeast Sir2 were clearly reported.
Based on the tandem affinity purification combining with the mass spectrometry technique, we previously identified several XRCC4 association proteins including SIRT2. Using the affinity precipitation technique, we corfirmed SIRT2 binds to XRCC4 in vivo. By serial deletion mutation analysis data, XRCC4(181-193) plays an important role in both SIRT2 and LigaseIV binding. From further point mutation experiments on the residues of this region, the lysine 188 shows a crucial role on SIRT2 binding. Binding of the nuclear XRCC4 and cytosoilc SIRT2 occurs in the prophase/prometaphase during mitosis. Other in vivo experiments concluded this binding may be attributed to the normal function of the G2/M DNA damage checkpoint and the spindle assembly checkpoint.
In order to further confirm the specificity and the binding affinity between SIRT2 and XRCC4, I overexpressed the His-tagged and GST-fusion proteins in bacterial system, purified them and use the surface plasmon resonance and the affinity precipitation techniques to perform binding assays. Data show that SIRT2 directly binds to XRCC4 and their binding behavior does not interfere the known binding of XRCC4 and LigaseIV. It therefore concludes a non-competing binding model.
In order to investigate whether the lysine 188 residue in the XRCC4 is chemically modified by the enzyme SIRT2 or not, I apply site-directed mutagenesis to alter this lysine to either arginine or glutamine, which mimicking the non-acetylating or fully acetylating of lysine residue. Data show the affinity is not altered whether the lysine residue is acetylated or not.
I also performed site-direct mutagenesis on the catalytic center, histidine 187, of the enzyme SIRT2. This residue is replaced by alanine or tyrosine to simulate the dead enzyme reserving protein-protein interaction function. From the data of binding assay, the affinity to XRCC4 of the mutant SIRT2(H187Y) and SIRT2(H187A) shows a certain level of decrease.
My reaserch shows that SIRT2 can directly and specifically binds to XRCC4 in vitro, and can directly form a complex with LigaseIV. From the binding assay performing on the mutants, I conclude that the binding of the SIRT2 and XRCC4 is belonging to a general protein-protein interaction, not an enzyme-substrate relationship. From the evidence obtained by tandem binding assay, it shows SIRT2 bind the pre-forming XRCC4-LigaseIV complex and form a stable complex structure.
Subjects
XRCC4
SIRT2
交互作用
interaction
Type
other
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