Figure 1
Subassemblies of MCMs form distinct, DNA unwinding-competent complexes with FACT. (A) Western blot analysis of HeLa whole cell extracts (left panel) and nuclear extracts (right panel), as well as the different immunocomplexes targeted by control (2B12), α hSpt16p (8D2), and α SSRP1 (10D1) mAbs. Immunoblotting was done using the indicated antibodies against pan-MCM or individual subunits. The amount of the Input is equivalent to 1/40 the IP. The identity of the protein band, marked by the asterisk, is unknown. The chart summarizes the constituents of the different FACT immunocomplexes. (B) Immunocomplexes were isolated as in (A) and subjected to DNA helicase assay. The reaction was conducted on a radiolabeled, 17-mer oligonucelotide annealed on the M13 single-stranded DNA ("HD", heteroduplex DNA). Upon protein removal, reaction mixtures were resolved by native gel. The locations of the annealed and displaced (ssDNA) substrates on the gel are shown. Displacement of the annealed substrate by heat denaturation is also shown (Boiled). (C) The DNA helicase activity of the FACT-MCM complex is ATP-dependent. The displacement of 17-mer oligonucleotide (SS) from the heteroduplex substrate (HD) by the 10D1-immunocomplex was assayed in the presence (lane 2) or absence (lane 3) of ATP, or in the presence of ATP-γ S (lane 4). (D) HeLa cell extracts were prepared from control (lanes 2 & 3), MCM4RNAi (lane 4), or MCM3RNAi (lane 5) cells. The displacement activity of the mock- (lane 2) or 10D1- (lanes 3-5) immunocomplex isolated from these extracts is shown.