Figure 7. Cooperative assembly of the CMG−Ctf4₃−Pol α-primase complex, and the demonstration that CMGs multimerized by Ctf4₃ have helicase activity and support DNA replication.

(a) Streptag-Ctf4 trimer on streptactin magnetic beads was added to either Pol α, CMG or a mixture of Pol α + CMG. Proteins were eluted with biotin and analyzed by SDS-PAGE (upper right). The assay was repeated in triplicate and gel scans were quantitated (below). The error bars show the standard deviation. The cooperativity is consistent with the stoichiometric assembly of 2(CMG–Pol ε)−1Ctf4₃−1Pol α-primsae while excluding most of the Ctf4₃ in a glycerol gradient analysis (Figure 7—figure supplement 1). The results are also consistent with negative stain EM of a mixture of CMG+Ctf4₃+Pol α-primase, showing the presence of a 2CMG−1Ctf4₃−1Pol α-primase complex (Figure 7—figure supplement 2). (b, c). Both CMGs in the 2CMG−Ctf4₃ factory are active. (b) Controls for testing CMG−DNA binding time in 0.1 mM ATPγS are in Figure 7—figure supplement 3. Native PAGE analysis of helicase assays upon preincubation of either CMG or CMG + Ctf4 for 2 hr with 0.1 mM ATPγS and a ³²P-forked DNA followed by 5 mM ATP to initiate unwinding. Timed aliquots were removed for analysis as indicated in the representative native PAGE gels. The plot represents results of triplicate assays. The mean value is indicated by the symbols and error bars show one standard deviation. (c) Preincubation experiments to determine the time for Pol α-primase to assemble onto the forked DNA are shown in Figure 7—figure supplement 4. Either CMG or CMG+Ctf4₃ were preincubated with ³²P-primed DNA fork and 0.1 mM ATPγS for 115 min, followed by addition of Pol α-primase and a further 5 min incubation before initiating replication/unwinding with 5 mM ATP and 0.1 mM each dNTP. Timed aliquots were removed for analysis as indicated in the representative native PAGE gels. DNAs having CMG bound enable Pol α-primase to extend the DNA to full length. Pol α-primase only extends to the forked junction on DNAs that lack CMG due to inability of Pol α-primase to perform strand displacement synthesis. The plot of full-length products represents results of triplicate assays. The mean value is indicated by the symbols and error bars show one standard deviation.

Figure 7—figure supplement 1. Densitometry analysis of CMGE−Ctf4₃−Pol α-primase.

Lane 10 of the SDS-PAGE of Figure 2—figure supplement 1 is shown to the left. The densitometry scan of the SDS gel lane 10 (right) was analyzed using ImageJ and indicates 2CMGE−1Ctf4₃−1Pol α-primase. The area of the Cdc45 peak was used as a proxy for CMG stoichiometry, and the area of the Cdc45 peak divided by the Cdc45 mw was assigned a value 2.0 because molar areas of Pol1, Pol12, and Ctf4₃ were about half the molar value of Cdc45. The amount of Ctf4₃, which overlaps Mcm4, was determined in two steps. First, the area of the Mcm2-7 region was divided by the molecular weight of Mcm2-7. The difference in area was deduced to belong to Ctf4₃, and calculated based on the molecular weight of a Ctf4 trimer. Calculated values are shown under the peaks. The stoichiometry approximates to 2 Cdc45 (and thus 2 Mcm2-7), 1 Ctf4₃, 1 Pol α, and 2 Pol ε.

Figure 7—figure supplement 2. EM observations of a 2CMG−Ctf4₃−1-Pol α-primase complex.

(a) 2D class averages of negatively stained EM images of CMG plus Ctf4 trimer indicate a 2CMG−Ctf4₃ complex. (b) The top panels are 2D class averages of negatively stained EM images of a mixture of CMG, Ctf4 trimer and Pol α-primase, which we interpret as a complex of 2CMG−Ctf4₃−1Pol α-primase. The bottom panels explain the interpretation of the images in the top 2D averages of panel b using yellow to color CMGs, blue to color the Ctf4 trimer, and green to color the Pol1 subunit of Pol α-primase. See text for details.

Figure 7—figure supplement 3. Establishing preincubation conditions for CMG binding to DNA before adding ATP for unwinding in helicase assays.

(a) Scheme of the assay (top) and native PAGE gel analysis (bottom) of CMG helicase activity at different times of preincubating DNA with CMG and 0.1 mM ATPγS, followed by adding 5 mM ATP to initiate unwinding. (b) Quantitation of the gels in panel (a). Minutes of preincubation of CMG, DNA and ATPγS are shown to the right of each line.

Figure 7—figure supplement 4. Establishing preincubation time for Pol α-primase binding to ³²P-primed DNA for replication with CMG+/-Ctf4.

(a) Top: scheme of the assay. Reactions containing 20 nM CMG + /- 10 nM Ctf4₃ were preincubated with 0.5 nM ³²-P primed fork DNA and 100 μM ATPγS for a total of 2 hr, and Pol α-primase was added at the times indicated, followed by a 10 min pulse of replication upon using 5 mM ATP and 100 μM each dNTP. Bottom: Reactions were quenched and analyzed by denaturing PAGE. Pol α-primase alone is unable to perform strand displacement, and therefore ³²P-primed sites on DNA forks lacking CMG are only extended to the forked junction. (b) Quantitation of the full-length products.