Figure 5.

Binding of Orp4 protein to the ars1 replication origin. Cell extracts were prepared from haploid cells expressing full length Orp4p (F), the N-terminal domain of Orp4p (N), or the C-terminal domain of Orp4p (C). Extracts from cells containing vector alone (V) served as a control. After SDS/PAGE the cellular proteins were transferred to a nitrocellulose membrane and subjected to a renaturation protocol. The membranes were incubated with a radiolabeled fragment of the ars1 replication origin in the presence or absence of a 100-fold excess of nonradioactive competitor DNA. The competitor DNAs consisted of the identical ars1 fragment (specific) or an unrelated fragment of similar size (nonspecific). The positions of full-length Orp4p, the N-terminal domain and the C-terminal domain are indicated as F, N, and C, respectively. (The expression of all three proteins was verified by Western blot analysis.) The position of endogenous Orp4p is indicated by E.