Abstract
Regulation of XRCC4 protein in DNA double strand break repair
DNA double strand break (DSB) is considered the most fatal type of DNA damage induced by radiation. Eukaryotic organisms equipped two major DSB repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination. We focused on XRCC4 protein, which binds to and cooperates with DNA ligase IV in the joining of DSB at the end stage of NHEJ. XRCC4 protein undergoes a number of post-translational modifications, including phosphorylation by DNA-PK. We guess that NHEJ is regulated through post-translational modifications. In order to reveal these modification mechanisms, it is necessary to evaluate the function of XRCC4 mutant, especially in terms of radiosensitivity. However, colony formation assay following cloning takes one or two month to get the experiment result. We sought to establish a rapid assay using fluorescent reporter protein.
A series of XRCC4 point mutants were created by PCR and then integrated into pIRES-DsRed vector, which drives expression of XRCC4 and DsRed from a single mRNA. These vectors were introduced into XRCC4-defective murine leukemia cell line M10. After irradiation, cells expressing XRCC4 with normal function would predominate because of increased survival. We can identify and measure XRCC4-expressing cells by fluorescence using flow cytometer.
