FIGURE 5.

Cells expressing sld2-m1,4 exhibit premature GINS-Mcm2-7 interaction in G₁, and cells expressing sld2-DNA exhibit no GINS-Mcm2-7 interaction. A, sld2-td cells expressing SLD2-wt, sld2-m1,4 (K416E,R435E,R436E), or sld2-DNA (K44E,K50E,K438E) at the restrictive temperature (37 °C) were arrested with α-factor and then released into HU for the indicated times. A–C, left panel) Whole cell extracts were analyzed by Western for expression indicated proteins. A, right panel, cell extracts were immunoprecipitated with antibodies directed against Sld2, followed by Western analysis for Sld2 or Mcm2. B, right panel, cell extracts were immunoprecipitated with antibodies directed against Psf2–5flag (a component of GINS), followed by Western analysis for Psf2, Mcm2, or Cdc45. C, right panel, cell extracts were prepared to isolate loaded Mcm2-7 complexes as described (29), using antibodies directed against Mcm4–9myc, followed by Western analysis for Mcm2, Psf2, Cdc45, or Sld2. D, model for assembly of CMG complex in wild-type yeast cells.