1. Mcm10 Regulates the Stability and Chromatin Association of DNA Polymerase-α 
Robin M. Ricke, Anja-Katrin Bielinsky 
Molecular Cell 
Volume 16, Issue 2, Pages (October 2004)
DOI: /j.molcel

2. Figure 1
Mcm10 Association with Replication Origins Is Cell Cycle Regulated and Requires the Mcm2-7 Complex
(A) ChIP was performed on YK10MYC cells (MCM10-9Myc) (Homesley et al., 2000) that were asynchronous or arrested in G1, S, or G2 phase. Extracts were prepared from formaldehyde-treated (+Formaldehyde) and untreated (−Formaldehyde) cells. Mcm10 and Orc2 were immunoprecipitated. PCR products derived from immunoprecipitated samples representing ARS305, ARS1, ARS603, ARS1004, and the nonorigin region R11 were visualized on agarose gels.
(B) ABy001 (mcm4-td, MCM10-9Myc) cells were arrested with nocodazole (Noc) at room temperature (RT) for 2 hr and shifted to 37°C in the presence of galactose with nocodazole for 1 hr. The culture was split and released at 37°C in the presence of galactose or released at RT in the presence of α factor for 3 hr. Flow cytometry indicated the DNA content of the cells throughout the course of the experiment.
(C) Western blot analysis of Mcm4-td, Mcm10-9Myc, and histone H3 on whole-cell extracts during the course of the experiment in (B).
(D) ChIP was performed as described in (A) on formaldehyde-treated cells grown according to the scheme in (B).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Mcm10 Migrates with the Replication Fork
(A) YK10MYC cells (MCM10-9Myc) were synchronized in G1 and released at 20°C. Samples were taken at the times indicated. ChIP was performed using anti-Myc and anti-Orc2 antibodies. PCR products representing ARS305, ARS1, ARS603, and ARS1004 and the nonorigin regions R11, ARS1 + 3 kb, and ARS kb were visualized on agarose gels.
(B) Flow cytometry indicated the DNA content of samples in (A).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Mcm10 Association with Chromatin Changes with the Cell Cycle
(A) YK10MYC cells (MCM10-9MYC) were synchronized in G1 and released at 22°C. Samples were taken at the times indicated. DNA content was monitored using flow cytometry.
(B) The histone association assay was performed on cells released from α factor as shown in (A). Whole-cell extracts (INPUTS) and precipitated proteins (Histone H3 IP) were analyzed by immunoblot. Mcm10-9MYC, Orc2, and histone H3 were detected.
(C) ABy029 cells (MCM10-9MYC, CDC45-3HA) were blocked in G1 (α), S (HU), or G2 (Noc.) and the histone association assay was performed. Mcm10-9Myc, Cdc45-3HA, histone H2B, and histone H3 were detected.
(D) Asynchronous YK10MYC cells (MCM10-9MYC) were subjected to the histone association assay under quantitative conditions. Fifty or one hundred microliters of extracts was immunoprecipitated with anti-histone H3 antibody. The amount of histone H3 in the supernatant (SN) and pull-down (PD) was compared.
(E) DNA present in the pull-down (PD) or supernatant (SN) fractions as prepared in (C) was purified and visualized on EtBr-stained agarose gels.
(F) The amount of chromatin-associated Mcm10-9Myc was estimated using 50 μl of crosslinked extracts in a histone H3 IP. Equal amounts of histone H2B, histone H3, and Orc2 were present in the input (INPUT) and pull-down (PD) fractions. Ratios of PD/INPUT for histone H2B, histone H3, and Mcm10-9Myc normalized against Orc2 are indicated.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Mcm10 Physically Interacts with the Polα-Primase Complex and RPA
(A) ABy003 cells (MCM10-9MYC, CDC17-3HA) were blocked in S phase with HU. WCEs were immunoprecipitated with anti-HA (12CA5), anti-Myc (9E11), or IgG in the presence or absence of DNaseI, as indicated. Mcm10-9Myc and Pri2 were detected with anti-Myc antibodies (9E11) and Pri2 antibodies, respectively.
(B) ABy003 cells were blocked in S phase with HU. Immunoprecipitations were performed with anti-histone H3 (H3), anti-Myc, and IgG in the presence or absence of DNaseI. Mcm10-9Myc, exonuclease I (ExoI), histone H2B, and histone H3 were detected by Western blot.
(C) ABy003 cells (MCM10-9MYC, CDC17-3HA) were synchronized in S phase with HU and immunoprecipitated with anti-Rpa2 (9H8), anti-Myc (9E11), or IgG, as indicated. Mcm10-9Myc and Rpa2 were detected with anti-Myc (9E11) and anti-Rpa2 (9H8), respectively.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Mcm10 Is Required for S Phase Progression
(A) Experimental design to examine the requirement for Mcm10 during DNA replication. At 37°C, cells were incubated in the presence of galactose to induce Ubr1 expression (Labib et al., 2000).
(B) ABy011 (GAL-UBR1, CDC17-3HA), ABy001 (GAL-UBR1, CDC17-3HA, mcm4-td), and ABy006 (GAL-UBR1, CDC17-3HA, mcm10-td) cells were treated as indicated in (A). DNA content was analyzed throughout the course of the experiment using flow cytometry.
(C) (Left) ABy011 and ABy006 cells were crosslinked with formaldehyde. WCEs were sonicated to fragment chromatin and heat denatured to reverse crosslinks. (Right) ABy011 and ABy006 samples were subjected to the histone association assay. Cdc17-3HA, Mcm10-td, Orc2, and histone H3 were detected by Western blot. Mcm10-td contains an HA epitope tag and was detected with an HA-specific antibody (16B12).
(D) ABy022 (CDC45-3HA, GAL-UBR1, MCM10+) and ABy023 (CDC45-3HA, GAL-UBR1, mcm10-td) cells were synchronized in G1 with α factor and released into hydroxyurea (HU) to block cells in S phase at room temperature (RT) or at 37°C in the presence of galactose. The chromatin association of Cdc45-3HA, Mcm10-9Myc, histone H3, and Pri2 were analyzed using the histone association assay.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Mcm10 Controls the Stability of Polα
(A) Asynchronous cultures of YKL83 (MCM10+, CDC17+, GAL-UBR1), ABy011 (MCM10+, CDC17-3HA, GAL-UBR1), and ABy006 (mcm10-td, CDC17-3HA, GAL-UBR1) at room temperature (RT) were shifted to 37°C in the presence of galactose for 90 or 180 min. Cdc17-3HA, Mcm10-td, Mcm7, and histone H3 were detected by Western blot. Numbers below the blots indicate the amount of Cdc17 normalized to histone H3.
(B) Asynchronous cultures of W303 (MCM10+, CDC17+), OAy644 (MCM10+, CDC17-3HA), and ABy013 (mcm10-1, CDC17-3HA) grown at room temperature (RT) were shifted to 37°C for 90 and 180 min. Histone H3 and Cdc17-3HA were analyzed by Western blot.
(C) An asynchronous culture of ABy018 (mcm4-td, CDC17-3HA, GAL-UBR1) grown at room temperature (RT) was shifted to 37°C in the presence of galactose for 90 or 180 min. Mcm4-td was detected by Western blot.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 Recruitment of Mcm10 and Polα-Primase to Replication Origins
Mcm10 exists in two cellular pools. Part of Mcm10 is associated with polα-primase. Mcm10 binds licensed replication origins at the end of G1 in an Mcm2-7-dependent manner at the same time as Cdc7-Dbf4 (Wohlschlegel et al., 2002). This allows for the recruitment of Cdc45 (Sawyer et al., 2004) and GINS and results in DNA unwinding and RPA loading. Assembly of Mcm10/polα-primase complexes onto DNA requires RPA.
Molecular Cell  , DOI: ( /j.molcel )