Rad51 and Rad52 aid in CENP-A^CaCse4 recruitment.

<p>(A) Standard ChIP assays followed by quantitative real time PCR (qPCR) were performed in wild-type, <i>rad51</i> or <i>rad52</i> for CENP-A^CaCse4-Prot A for <i>CEN5</i> and <i>CEN7</i>. qPCR amplification from a non-centromeric (<i>non-CEN</i>) control was also performed to detect the background DNA elution in the ChIP assays. Enrichment of CENP-A^CaCse4 at the centromeres was calculated as a percentage of the total chromatin input and values were plotted as mean of three independent ChIP experiments ± SD. (B) Western blot analysis performed with the whole cell lysates from wild-type and homologous recombination (HR) and non-homologous end joining (NHEJ) mutants using anti-CENP-A^CaCse4 antibodies. PSTAIRE was used as a loading control. The relative levels of CENP-A^CaCse4 (CENP-A^CaCse4/PSTAIRE) was computed for each mutant and plotted in a bar graph. (C) Co-immunoprecipitation assays for the two sets of strains carrying Rad51-V5 and Rad52-V5 were performed using anti-V5 antibodies. Precipitates were analyzed by western blotting with anti- CENP-A^CaCse4 antibodies. In each case, untagged strains and no (-) antibody fractions were used as controls. Blue asterisk indicates a non-specific band.