Figure 6.

(See previous page). RecQ4-dependent assembly of replisome components, initiation of replication, and unwinding of dsDNA. (A) RecQ4-dependent and -independent binding of initiator proteins to the plasmids. The bead-coupled plasmids were incubated in mock- or RecQ4-depleted HSS at 23°C for 30 min to allow pre-RC assembly. Subsequently, 2 vol of mock-depleted NPE with or without 1 μM p21, or RecQ4-depleted NPE with or without 530 nM N-terminal fragment of RecQ4 (N2), was added to 1 vol of each corresponding HSS and further incubated for 30 min. Proteins bound to the plasmids (plasmid) or in the extracts (extract) were analyzed by immunoblotting. (B) RecQ4 promotes replication of the plasmids in NPE. The bead-coupled plasmids (3.0 kbp) assembled with pre-RC were incubated in NPE as shown in Figure 6A, except for the presence of [α³²P]-dATP. The reactions were terminated at the indicated time and the incorporation of [α³²P]-dATP into DNA was detected after separating DNA by agarose gel electrophoresis. (C) RecQ4-dependent and -independent recruitment of replication proteins to the site-specific origin. The bead-coupled plasmids (7.6 kbp) bound with GAL4-Cdc6 were incubated at 23°C for 30 min in Cdc6-depleted or Cdc6 and RecQ4 double-depleted HSS. After the incubation, mock-depleted or RecQ4-depleted NPE containing 1 ng/μl aphidicolin was added to the corresponding HSS and further incubated for 30 min. The localization of proteins bound to the plasmids was examined by ChIP analysis as described in Figure 2, except for using primer sets at X and Z, proximal and distal to the origin, respectively. Antibodies used for ChIP are indicated at the top of each panel. (D) Stable binding of Cdc45, Mcm2-7, and GINS to the plasmid both in the presence and absence of RecQ4. The bead-coupled plasmids were incubated in mock- or RecQ4-depleted extracts as described in Figure 6A. After incubation in the mock- or RecQ4-depleted NPE, plasmid-beads were isolated and washed with the buffer containing various concentrations of NaCl. Proteins bound to the plasmids (plasmid) and in the extracts (extract) were analyzed by immunoblotting. (E) RecQ4 promotes unwinding of DNA in NPE. The bead-coupled plasmids (7.6 kbp) were incubated at 23°C for 30 min in RecQ4-depleted HSS, and then 2 vol of RecQ4-depleted NPE was added with and without a 720 nM N-terminal fragment of RecQ4 (N2) in the presence of 67 ng/μl aphidicolin. After 30 min incubation, the plasmids were isolated and treated with P1 nuclease at 38°C for 10 min. Total DNA isolated from the mixture was further digested with BamHI, and the purified DNA was separated by agarose gel electrophoresis. As a positive control, mock-depleted extracts were used for the unwinding assay (mock).