Purification, characterization, and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein.
The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved in the repair of DNA single-strand breaks. To understand how these BRCT regions specify protein-protein interactions and contribute to DNA repair function, we have overexpressed and purified the distal BRCT domain of XRCC1 with the goal of structure determination. The cDNA encoding this BRCT region (X1BRCTb) was inserted into the pET29 bacterial expression vector; the polypeptide was expressed in mostly soluble form and then purified by anion-exchange and gel filtration chromatography. Crystallization screening with the purified material resulted in the formation of large bipyramidal crystals. Crystals formed within several hours at room temperature from salt solutions of ammonium sulfate. Crystals diffract to approximately 2.85 A and were found to be in space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit cell dimensions a = 100.43 A, c = 105.62 A. Crystals of similar character have also been obtained after incorporation of selenomethionine during expression of the protein. Efforts are now under way to determine the molecular structure of the X1BRCTb domain. These studies are likely to give insight into the interaction between XRCC1 and DNA ligase III and into general structural features of BRCT domains that exist in many other proteins.
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550, USA.
K
K
Thornton
M
M
Forstner
M R
MR
Shen
M G
MG
West
B
B
Rupp
M P
MP
Thelen
D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011487:Protein Conformation; D000076105:X-ray Repair Cross Complementing Protein 1
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human