Figure 4: Replisome displacement of a single nucleosome.

(a) Experimental configuration. Leading strand replication was carried out using the T7 replisome on a Cy5-labelled parental template containing a single nucleosome with minimal dsDNA downstream. (b) Nucleosome transfer after replication. The replication product was exonuclease III-digested and assayed on a denaturing gel (lane 7). Lane 1 is a ladder and lanes 2–6 are control experiments. Lanes digested with exonuclease III were loaded with five times as much sample as the other lanes to achieve more accurate quantification. N=4 replicates (Supplementary Fig. 7). (c) A line scan of lane 7 contained contributions from both the transferred nucleosome as well as background. In particular, a fraction of replisomes did not proceed past the nucleosome, and another fraction contained inactive replisome bound at the initial fork (see lane 6). The background in lane 6 is removed from lane 7 during subsequent analysis. The template schematic right of the line scan explains some features of the band positions in the gel and the line scans.