Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation

Abstract

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.

Figure 1.

Alignments of Mcm10 and DmMcm10 complements ScMcm10. (A) Mcm10 from Homo sapiens, X. laevis, D. melanogaster, and S. cerevisiae all share conserved core (black) and zinc finger motif (shaded box). Also shown is shared conserved region among metazoans (stippled). Line through at amino acid 95 of DmMcm10 represents C-terminal fragment able to complement ScMcm10. (B) DmMcm10 supports growth of S. cerevisiae strain null for ScMcm10. Null strain with ScMCM10 pRS316 URA3 plasmid grows on CM-URA (bottom right) but not on CM + 5-FOA (top middle). Transformation of null strain with Dm- MCM10 pRS315 Leu2 results in ability to grown on CM + 5-FOA (top middle). When taken from 5-FOA and plated on CM-Leu growth is observed (bottom left) but not observed on CM-Ura (bottom right).

Figure 2.

Mcm10 interacts with members of the pre-RC and Hp1. (A) Interactions between Mcm10::GFP and Mcm2, Dup, Cdc45, Orc2, Hp1, and Mcm10, respectively. Extracts from KC cells induced or not induced to produce Mcm10::GFP were immunoprecipitated with anti-GFP. Precipitate was loaded onto SDS-PAGE gel and probed with antibodies indicated. No interactions were detected for Orc1 and Mcm5. (B) Western blot probed with antiMcm10 of immunoprecipitation from embryo extracts with antibodies against Cdc45, Dup, Mcm2, Orc2, and Hp1 respectively. Mock was performed with preimmune rabbit IgG. Input lanes in A and B represent 4% of total protein. (C) Western blots of extracts from KC cells probed with antibodies against Mcm2, Cdc45, Mcm5, Orc1, Mcm10, Dup, and Hp1, respectively.

Figure 4.

Effects of depletion of Mcm10 and Orc2, respectively, on cell growth and DNA content. (A) Cell density >6 d of KC cells either untreated or incubated with specific dsRNA to Mcm10, Orc2, and Dock, respectively. (B) Cumulative cell divisions >18 d in cells incubated with specified dsRNA or untreated. Cells were diluted every 3 d to 1 × 10⁶cells/ml and reinoculated with specific dsRNA. Data points based on replica hemacytometer counts and calculations of doubling time before each dilution. (C) Cells from B were collected after 10 cell cycles, fixed, RNase treated, and stained for FACS analysis.

Figure 3.

RNAi depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2. (A–E) Specific dsRNA added at final concentration of 10 nM to KC cells as indicated at top of panels and depletions monitored by Western blots >5 d. Blots probed with antibodies as indicated to left of panels.

Figure 6.

Chromosome condensation defects in KC cells depleted of replication proteins. Micrographs of metaphase KC cell chromosomes stained with Hoechst. Metaphase chromosomal defects for each RNAi depletion after 10 cell cycles were sorted into categories: I, II, and III for defects in lateral condensation, and I′, II′, and III′ for fragmentation. Representative chromosomes for wild type (A), Dock depleted (B), Mcm10 depleted (C), Cdc45 depleted (D), Mcm2 depleted (E), Mcm5 depleted (F), and Orc2 depleted (G). Bar, 5 μm.

Figure 5.

Relative stability of Mcm10, Cdc45, Mcm2, and Orc2 in KC cells after respective RNAi treatments. Cells after 10 cell cycles in presence of indicated dsRNA species were harvested and extracted. Extracts were run on SDS-PAGE gels and resulting blots probed with indicated antibodies.