Figure 2.
Chromatin Inhibits DNA Replication In Vitro
(A) Reaction scheme for replication reactions and CMG recruitment on 5.8-kb circular bead-bound ARS1-containing templates using S phase extract or purified proteins. Chromatin assembly and MCM loading were performed as in Figure 1A. Loaded MCMs were further phosphorylated with DDK before they were added to an S phase extract or purified replication proteins.
(B) CMG recruitment on naked DNA and on chromatin in the presence of purified initiation and replication factors (Sld3/7, Cdc45, Dpb11, Polε, GINS, Sld2, Mcm10, and S-CDK). Reactions were performed as in Figure 3A. Beads were collected and washed with 0.3 M KCl. Recruitment of the CMG with or without DDK was assessed by immunoblotting using antibodies recognizing Cdc45, Psf1 (GINS), and Mcm7 (MCM) subunits.
(C) Replication reactions on naked DNA and on chromatin conducted as shown in (A) using the minimal replication system (Yeeles et al., 2015). In this and all subsequent replication reactions, DNA was visualized by incorporation of [α32P] deoxycytidine triphosphate (dCTP) into nascent DNA and products were separated through a 0.7% alkaline agarose gel.
(D) Replication reactions on naked DNA and on chromatin using the complete replication system as described in Yeeles et al., 2016.
(E) CMG recruitment in S phase extract on either naked DNA or chromatin in the presence and absence of DDK phosphorylation.
(F) Replication reactions on naked DNA compared to chromatin using S phase extract in the presence or absence of DDK.