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PLoS One
2007 Sep 05;29:e833. doi: 10.1371/journal.pone.0000833.
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Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells.
Crevel G
,
Hashimoto R
,
Vass S
,
Sherkow J
,
Yamaguchi M
,
Heck MM
,
Cotterill S
.
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BACKGROUND: The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.
METHODOLOGY/PRINCIPLE FINDINGS: We performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95-99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.
CONCLUSIONS/SIGNIFICANCE: These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells.
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17786205
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Figure 1. Dpa1 mutants show abnormal chromosome condensation and unorganised mitotic spindles.Neuroblasts of heterozygous or homozygous third instar larvae (prepared as in 4) were stained for: centrosomes (red) using CP190 antibodies (a kind gift from Will Whitfield) and Goat anti-rabbit TxRed (Jackson labs); spindles (green) using anti-tubulin (Clone DM1A-Sigma) and goat anti-mouse Alexa 488 (Jackson Labs); and DNA (blue) using DAPI (Sigma). Heterozygous neuroblasts showed normal spindles with correct localization of CP190 at the poles in metaphase (A/B) and anaphase (B), and normally condensed chromosomes. Homozygous neuroblasts (C/D) showed fragmented, hyper-condensed and aneuploid chromosomes, and disorganised mitotic spindles usually lacking CP190.
Figure 2. a. Schematic detailing the regions of each MCM targeted for dsRNA.Base positions are quoted relative to the start AUG. Shaded arrows designate the 2nd dsRNA made from MCM6/7. b. Agarose gel of RT-PCR products showing the effects of RNAi depletion of individual MCMs on MCM mRNA expression levels. Horizontal numbers designate the MCM targeted by RNAi. Vertical numbers show the RT PCR target. The data presented is one complete representative data set from multiple repetitions. c. Western blot to show the effect of RNAi depletion of individual MCMs on MCM protein expression levels in total cell extracts. Horizontal numbers designate the MCM targeted by RNAi. Vertical numbers show the antibody used for western blotting. Tubulin expression is included as a loading control. The data presented is one complete representative data set from multiple repetitions.
Figure 3. Immunoblot to show the effect of MCM depletions on the levels of MCM2, MCM5 ORC5 and CDC45 bound to chromatin.Coomassie stained histones are included as a loading control. The amount of each protein was compared to the histones using Alpha Innotech imager software. Horizontal numbers show the MCM protein targeted by RNAi. Several repetitions of this experiment were performed and this represents 1 complete data set.
Figure 4. Day 5 growth profiles of MCM targeted cells compared to a control cell line.The data presented shows combined data from seven independent experiments. The data is expressed as a percentage of the control. Lane 1 is the control and lanes 2-8 are MCM 2-8 resepectively.
Figure 5. FACS analysis profiles of cells targeted with dsRNA against MCM2-7 proteins as compared to control targeted cells.The data presented is one complete representative data set from multiple repetitions.
Figure 6. a. Agarose gel of RTPCR products to show the effects of RNAi depletion of MCM5 and MCM8 and MCM 5 plus 8 on the MCM5 and 8 levels.b. FACS profiles of cells targeted with dsRNA against MCM8, and MCM5 plus MCM8. The data presented is one complete representative data set from multiple repetitions.
Figure 7. Immunoblot to show the effect of MCM depletions on the levels of PCNA bound to chromatin.Coomassie stained histones are included as a loading control. The amount of PCNA was compared to the histones using Alpha Innotech imager software. Horizontal numbers show the MCM protein targeted by RNAi.
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