Fig. 5: UBP expression, binding profile and the role of the ucm in vivo.

a Western blot (upper panel) of a titration series of purified recombinant UBP (lanes 1–5) and whole cell extract from known numbers of cells (lanes 6–9). The lower panel indicates a graph of signal intensity versus UBP quantity with a linear curve fit applied. The blue line indicates a signal intensity for UBP in extract from 1.7 × 10⁷ cells, which is clearly in the linear range, and an extrapolation to the x-axis to reveal the corresponding amount of UBP. b Growth, stationary and death phases of a S. islandicus culture monitored by OD6000 nm. Western blotting of whole cell extracts at the indicated timepoints with antisera to UBP and, as a loading control, the general transcription factor, TBP. c Upper panel: chromatin precipitation experiments with anti-sera against UBP and MCM in the wild-type and M7-mutation-containing strain. qPCR was performed with primer pairs specific for oriC1 and oriC3, the bar graphs indicate the mean of three independent experiments with the values of recovery of input-normalized oriC1 DNA divided by that of input-normalized oriC3 DNA. Individual data points are shown as diamonds. ChIP results from the wild-type strain are shown in white, from M7 in pale blue. The lower panel shows ChIP experiments using anti-Orc1–1 antisera in the WT, M7 and M12 strains, Values are normalized to input DNA, as above; the bars represent the mean of three independent experiments, with individual data points shown as diamonds. d A genome-wide profile of UBP binding assayed by ChIP-Seq for wild-type and M7 mutant strains, the positions of the three replication origins are indicated in red. e Enlarged view of the oriC1-containing region of the chromosome for the data shown in panel (d). f Aggregated analysis of the relative abundance of UBP ChIP signal in regions up to 200 bp upstream of open-reading frames (ORFs), across the ORFs themselves and in the downstream 200 bp. g A consensus sequence was identified using the MEME suite²⁶ using a sequence library of the top 100 ranked UBP sites identified by the MACs peak caller.