Skip to main content
. 2020 Jul 23;9:e58571. doi: 10.7554/eLife.58571

Figure 2. The Mcm2 NTE is important for DNA replication.

(A) Experimental outline. (B) In vitro DNA replication reactions were performed on naked (lanes 1–3) or chromatinized (lanes 4–6) circular plasmid DNA (p1017, 4.8 kbp). TEV protease was added to each reaction following MCM loading for 1 hr at 30°C, before addition of DDK and standard initiation/replisome factors. Chromatin replication reactions additionally contained FACT and Nhp6. Products were analyzed by 0.8% denaturing agarose gel-electrophoresis and autoradiography. Lead: Leading strand product; lag: Lagging strand product. (C) Purified Cdt1·MCM complexes containing either wildtype Mcm2 (Cdt1·MCM2-WT), Mcm2-TEV (Cdt1·MCM2-TEV), or Mcm2-2A (Cdt1·MCM2-2A).

Figure 2—source data 1. Figure 2A.
Figure 2—source data 2. Figure 2C.

Figure 2.

Figure 2—figure supplement 1. FACT/Nhp6-dependent chromatin replication.

Figure 2—figure supplement 1.

(A) Purified FACT and Nhp6. Samples were analyzed by SDS-PAGE and Coomassie stain. (B) In vitro DNA replication reaction was performed on chromatinized p470 (10 kbp) in the absence or presence of FACT and Nhp6 as indicated. Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography.
Figure 2—figure supplement 1—source data 1. Figure 2—figure supplement 1A.
Figure 2—figure supplement 1—source data 2. Figure 2—figure supplement 1B.
Figure 2—figure supplement 2. Attenuation of DNA synthesis in the presence of Mcm2-TEV is dependent on TEV protease cleavage.

Figure 2—figure supplement 2.

Standard DNA replication reactions using naked or chromatinized p1017 (4.8 kbp) as template were performed with Cdt1·MCM2-TEV or Cdt1·MCM2-WT as indicated. TEV protease or mock buffer was added to the reaction following MCM loading and preceding origin activation as indicated. Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography.
Figure 2—figure supplement 2—source data 1. Figure 2—figure supplement 2.