Accumulation of P-H2A in BY4741 (wt) and Y11310 (asf1Δ) strains grown with 0.05% (+) or without (−) MMS for 2 h. Total and Ser 129-phosphorylated histone H2A were detected by western blot as described previously (Downs et al, 2000).

Frequency of homologous recombination in chromatin assembly mutants. (A) Plasmid pRS316sU carrying the inverted repeat system SU and its recombination product. Grey boxes indicate the inverted repeats. (B) Frequency of Leu+ recombinants in strains BY4741 (wt), Y11310 (asf1Δ), Y05437 (cac1Δ) and BYac1-1B (asf1Δ cac1Δ).

Accumulation of Rad52–YFP foci in asf1Δ cells. Rad52–YFP foci were visualized by fluorescence microscopy in asynchronous cultures of BY4741 (wt) and Y11310 (asf1Δ) cells transformed with plasmid pWJ1344. (A) Fluorescence and brightfield microscopy of asf1Δ cells expressing the Rad52–YFP fusion protein. (B) Percentage of unbudded (G1) and budded (S and G2/M) wild-type and asf1Δ cells containing Rad52–YFP foci. The total numbers of analysed cells were 55 and 199, and 62 and 396 for unbudded and budded wild-type and asf1Δ cells, respectively.

Effect of asf1Δ on plasmid topology and chromatin structure in strains BY4741 (wt) and Y11310 (asf1Δ). (A) Topoisomer distribution of plasmid pRS316sU. (B) MNase accessibility of bulk chromatin and (C) nucleosome positioning in the SU recombination system. r and sc, relaxed and supercoiled plasmids, respectively; MN, MNaseI. A scheme of the SU region analysed is shown. The asterisk indicates an MNase cleavage site protected in asf1Δ versus wild type.

Physical analysis of spontaneous and HO-induced SCE in WS (wt) and WS-asf1 (asf1Δ) strains. (A) Plasmid pRS316-TINV carrying the inverted repeat system TINV and the dimer resulting from unequal SCE between the inverted repeats. (B) Kinetics of HO-induced SCE. Mid-log-phase cells from SC–3% glycerol/2% lactate plus 5 μg/ml doxycycline were grown for different times in SC–2% galactose (Gal). Spontaneous SCE in the absence of HO breaks is observed as the accumulation of the 2.9-kb and 4.7-kb bands in asf1Δ cells in glycerol-lactate (t=0). The quantification of the HO-induced DSB (HOs 2.4 kb) and the 4.7-kb recombination product (REC 4.7) is shown. The average of two experiments performed with independent transformants is plotted. Variation for each point was <30%. DNA was isolated, digested with XhoI–SpeI and analysed by Southern blot with the ClaI–EcoRV LEU2 internal fragment for spontaneous and HO-induced SCE. Bands resulting from HO cleavage (HO-S and HO-X) and recombination intermediates (REC bands) were quantified in a Fuji FLA3000 and normalized relative to the total plasmid DNA. The asterisk shows unspecific hybridization.