Fig. 8: Effect of the A62S mutation on the DNA binding properties of Dpb3–Dpb4 and Dls1–Dpb4 heterodimers.

a Representation of Dpb4, Dpb3, and Dls1 proteins. The histone fold domain (HF) is shown. b 3D structure of Dpb4–Dpb3 and Dpb4–Dls1 heterodimers. The histone fold domain of Dpb4 is in orange, while that of Dpb3 and Dls1 is in light blue and green, respectively. A62 is shown as a red stick. The 3D structure of Dpb4–Dpb3 was extracted from PDB 6WJV³⁵, while the 3D model of Dls1 was built using I-TASSER⁹⁰. c HO expression was induced at time zero by galactose addition to G2-arrested cells that were kept arrested in G2 by nocodazole. Relative fold enrichment of Dpb4-HA and Dpb4-A62S-HA compared to untagged Dpb4 (no tag) was evaluated after ChIP with anti-HA antibodies and qPCR analysis. The mean values of three independent experiments are represented with error bars denoting s.d. ***p < 0.005 (unpaired two-tailed Student’s t-test). d Western blot with anti-HA antibodies of protein extracts from G2-arrested cells. The experiment was performed independently three times with similar results. e, f EMSA with a 61 bp dsDNA and increasing concentrations of Dpb3–Dpb4 and Dpb3–Dpb4^A62S (e), and Dls1–Dpb4 and Dls1–Dpb4^A62S (f) complexes. Bands corresponding to free DNA (F), and protein–DNA complexes with higher stoichiometry (asterisk) are denoted. The experiments were performed independently two times with similar results. g Model for Dpb4 function at DSBs. After DSB formation, the Dls1–Dpb4 dimer promotes the association of Isw2 to DSBs, which catalyzes nucleosome sliding/removal and facilitates MRX association to them. The Dpb3–Dpb4 dimer, possibly in complex with Pol ε, in turn, uses its histone chaperone activity to induce re-deposition/exchange of H3 and H4 histones (dark violet), whose subsequent H3 methylation by Dot1 (red dots) leads to H3 exposure to Rad9 recognition.