(A) Surface amino acid conservation of yeast Scc3. Residues in the DNA binding domain are well conserved. (B) DNA binding residues are located in three surface patches of Scc3. (C) DNA binding fluorescence polarization of 6-FAM labelled 32 bp dsDNA by variants of the Scc3-Scc1 subcomplex. Data points corresponding to the average of three independent experiments were fitted to a standard binding equation assuming a single binding site using Kaleidagraph. Standard deviations are depicted as vertical error bars. Apparent dissociation constants (KD) are noted below. (D) Tetrad analysis of diploid budding yeast strains expressing ectopic wild-type or mutant versions of Scc3 under control of the endogenous promoter in an SCC3/scc3Δ background (strains C5013, C5014, C5015, C5043, C5033). Images were recorded after three days at 30°C on rich media. Genetic marker analysis identified Scc3(mutant), scc3Δ cells (circles). (E) ChIP-qPCR analysis of binding to centromeric (cen), pericentromeric (pericen) or chromosome arm (arm) regions (chromosomes IV, V, and VI as indicated) of untagged (strain C3) or PK6-tagged wild-type or mutant versions of Scc3 expressed from an ectopic locus under its endogenous promoter (strains C5013, C5043, C5033). The fractions of immunoprecipitated DNA relative to input DNA are plotted as circles for two biological repeats with two technical repeats each (same colour pairs). Mean values of all four data points are shown as lines.