Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.
Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.
Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.Show MeSH
Related in: MedlinePlus
Mentions: To determine whether the radiosensitivity arose from a deficit in DSB repair per se, we again used a single-cell assay. We transfected with miRNA, irradiated and scored γ-H2AX foci, a marker of unrepaired DSBs, in cells expressing the EmGFP transfection marker. We irradiated at 0 or 1 Gy, then scored cells in various treatment groups at 0.5 h post-irradiation, to examine the ability to form γ-H2AX foci, and at 4 h post-irradiation, to examine the ability to recover. There was a low background of spontaneous foci in the non-irradiated cells (Figure 5). This increased to about 30 foci/per cell at 30 min following treatment with 1 Gy of 137Cs γ-rays, consistent with the predicted number of DSBs at this dose assuming a diploid mouse genome. The increase was similar in all four experimental groups. After 4 hours, most of the foci resolved in the wild-type genetic background (with or without PSPC1 knockdown). Most of the foci also resolved in the NONO-deficient cells transfected with control miRNA. However, dual deficiency in NONO and PSPC1 resulted in near absence of recovery at the 4-h time point. Taken together, results indicate that deficiency in NONO/PSPC1 function had no effect on the formation of γ-H2AX foci, which is a very early step in the cascade of events involved in DSB repair. Deficiency did affect the resolution of γ-H2AX foci, which is a very late step.
Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.