Figure 6.

B2 element is important when nucleosomes flank ARS1 DNA. A, reaction scheme for helicase loading assay performed in B. ORC-bound ARS1–DNA templates were either assembled into nucleosomes or not. These templates were tested for salt-stable helicase loading. DNA-associated proteins were detected by immunoblot. B, ARS1–DNA-associated Mcm2-7 and H2B amounts were detected by immunoblot. WT and B2- mutant ARS1 DNA are compared. C, comparison of origin-proximal nucleosome positioning for in vitro assembled ARS1 WT (red) and ARS1 B2- (blue) nucleosomal DNA templates. Origin-proximal nucleosome positioning was determined by high-throughput MNase-Seq of pUC19 ARS1-WT or pUC19 ARS1-B2- plasmids assembled into nucleosomes. Peaks represent the location of the midpoint of the protected nucleosomal DNA.