Fig. 7: Enhanced Arp5 chromatin binding and DNA accessibility during YCS.

a Apn1 and Ogg1 co-precipitate with actin. Bead-bound bicyclic peptides specific for F-actin (A18), F/G actin (A15)⁷⁵, or the control TATA2⁷⁵, were used to recover proteins from total yeast extracts. Proteins were visualized by silver staining, and prominent bands were identified by mass spectrometry from gel slices (see Methods, red = A15 and A18-pulldown, black = A15 only). The same fractions were analyzed by western blots for Apn1, Ogg1, Mcm2, and tubulin. Uncropped blots and mass spectroscopy results are in the Source data files. b Nuclear Apn1 levels drop slightly during YCS. APN1-GFP NUP49-RFP cells (GA-10504) cultured in SC were treated with DMSO ± 50 μg/ml Zeocin and 0.5 μM CMB4563 for 60 min. Spinning disc confocal images were captured of living cells in agarose plugs, and ImageJ quantified Apn1-GFP and Nup49-RFP intensities were plotted. Apn1-GFP was normalized to Nup49-RFP. Bar = 5 μm; n = cells imaged (n = 313, DMSO, and n = 763, Zeo + CMB). White bar = median; significance determined by Unpaired t test with Welch’s correction, two-tailed; p < 0.0001. c INO80 subunit Arp5, but not Apn1, is slightly enriched on chromatin during YCS. APN1-9Myc tagged cells exponentially cultured in SC were treated with DMSO ± 50 μg/ml Zeocin and 0.5 μM CMB4563 for 80 min, and subjected to chromatin fractionation^72,75. Total (T), soluble (S), and chromatin pellet (P) fractions were probed on western blots for Myc (9E10), Arp5, Orc2, and tubulin (see “Methods”). Full blots and quantitation in Source Data files. d P/T values for Apn1-Myc and Arp5 normalized to Orc2 are plotted, 1 = DMSO control. Anti-Orc2 cross-reacts with a 5kDa-smaller cytosolic protein (*). Uncropped blots are in Source data files. e Dam accessibility assay⁷⁸ monitors the relative accessibility of GATC motifs to ectopically expressed methylase (sketch modified from⁹⁹). WT cells (GA-1981) carrying p415GAL (control) or p415GAL-Dam were treated with 50 μg/ml Zeocin, 0.5 μM CMB4563, or both for 80 min. Total genomic DNA was isolated, digested with DpnI, analyzed on a 1% agarose gel and stained by SYBR safe. DpnI-insensitive (intact genomic band) and DpnI-sensitive (below intact chromosomes) were quantified by ImageJ. Ratios from 5 biological replicas are plotted.