DSB-triggered hMSH5 chromatin association is disrupted by phosphorylation deficient mutant hMSH5^Y742F.

<p>(<b>A</b>) ChIP analysis of the effects of hMSH5^Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5^Y742F. Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5^Y742F and selected with 10 µg/ml blasticidin. (<b>B</b>) Expression of hMSH5 and hMSH5^Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5^Y742F cells. (<b>C</b>) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I-<i>Sce</i>I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. (<b>D</b>) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s <i>t</i>-test.