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United States Department of Agriculture

Agricultural Research Service

Title: Characterization of the Xrcc1-Dnaligase III Complex in Vitro and Its Absence from Mutant Hamster Cells

Authors
item Caldecott, Keith - LAWRENCE LIVERMORE LAB
item Tucker, James - LAWRENCE LIVERMORE LAB
item Stanker, Larry
item Thompson, Larry - LAWRENCE LIVERMORE LAB

Submitted to: Nucleic Acids Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 27, 1995
Publication Date: N/A

Interpretive Summary: The building blocks or genes of organisms are encoded in their DNA. Changes in the DNA causes alterations in the genes and leads to mutations. Some mutations are good while others are harmful or even lethal. Thus, how biological systems monitor their DNA and repair undesirable changes in their DNA is of basic biological and agricultural interest. For example, all attempts at improving farm animal breeds and crop plants is fundamentally linked to desirable changes in the genes of these animals and plants resulting in improved qualities. The mechanism controlling how organisms monitor and repair their DNA is highly complex and poorly understood. In this work a human DNA repair protein referred to as XRCC1 was experimentally produced allowing for production of an antibody to this important DNA repair protein. Antibody molecules are serum proteins and can be used as tags to label and tract other proteins, in this case to label and track the XRCC1-DNA ligase complex. The exact biological role of the XRCC1 complex in DNA repair is unknown. However, this study clearly demonstrates that the XRCC1 complex is much reduced in mutants that have only limited abilities to repair their DNA. The methods for experimental expression of XRCC1 along with the development of a high affinity monoclonal antibody will allow detailed examination of this complex and evaluation of its role in the DNA repair process.

Technical Abstract: The human DNA repair protein XRCC1 was over expressed as a histidine- tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating the Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western Blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the approximate 6-fold reduced DNA ligase III activity observed previously en EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (>10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.

Last Modified: 10/21/2014
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