Figure 3

Functional attributes of the two distinct FACT-MCM assemblies. (A) HeLa WCE was subjected to immunoprecipitation with a control (lane 2), 8D2 (lane 3), or 10D1 (lane 4) antibody. Lane 1 is the extract input for the IP (1/40). Existence of pre-RC components (ORC1 and Cdc6) and Cdc45 in these immunoprecipitates was detected by specific antibodies. (B) ChIP was performed as described in Methods. Sonicated chromatin fragments were prepared from cells at different stages: asynchronous (lanes 1, 4, 7 & 10), G₁/S (lanes 2, 5, 8 & 11), and G₂/M (lanes 3, 6, 9 & 12). Immunoprecipitation was done with either control (IgG, lanes 7-9), 8D2 (lanes 4-6, 10-12), or 10D1 (lanes 1-3) antibody. Products from final PCR analysis using primers specific to lamin B2 origin (lanes 1-9) or to a non-transcribed region (lanes 10-12) were resolved by 1.5% agarose gel. Precipitated products are shown in the upper panels, DNA input in the lower. (C) Degree of origin association of the two immunocomplexes was quantitatively determined by the normalized ratio of amplified origin sequence, with the ratio of the asynchronous immunoprecipitate represented as 1. The histograms summarize such calculation and compares the origin binding of the FACT immunocomplex (8D2, left; 10D1, right) at each cell cycle stage. Data are averaged ± standard deviations of three independent experiments. (D) ChIP was performed as in (B). Sonicated chromatin fragments were prepared from control, MCM3^RNAi, and MCM4^RNAi cells (HeLa). Immunoprecipitation was done with either 8D2 (top panel) or 10D1 (middle panel) antibody. Presence of origin fragment was detected by PCR. PCR products from input DNA are shown in the bottom.