Dynamic localization of the DNA replication proteins MCM5 and MCM7 in plants

Abstract

Genome integrity in eukaryotes depends on licensing mechanisms that prevent loading of the minichromosome maintenance complex (MCM2-7) onto replicated DNA during S phase. Although the principle of licensing appears to be conserved across all eukaryotes, the mechanisms that control it vary, and it is not clear how licensing is regulated in plants. In this work, we demonstrate that subunits of the MCM2-7 complex are coordinately expressed during Arabidopsis (Arabidopsis thaliana) development and are abundant in proliferating and endocycling tissues, indicative of a role in DNA replication. We show that endogenous MCM5 and MCM7 proteins are localized in the nucleus during G1, S, and G2 phases of the cell cycle and are released into the cytoplasmic compartment during mitosis. We also show that MCM5 and MCM7 are topologically constrained on DNA and that the MCM complex is stable under high-salt conditions. Our results are consistent with a conserved replicative helicase function for the MCM complex in plants but not with the idea that plants resemble budding yeast by actively exporting the MCM complex from the nucleus to prevent unauthorized origin licensing and rereplication during S phase. Instead, our data show that, like other higher eukaryotes, the MCM complex in plants remains in the nucleus throughout most of the cell cycle and is only dispersed in mitotic cells.

Figure 1.

Expression of the Arabidopsis MCM2-7 complex is coregulated during development. A, Quantitative RT-PCR analysis of MCM2-7 mRNA abundance in Arabidopsis vegetative (shaded green) and floral (shaded blue) tissues and in suspension cell culture (shaded yellow). The tissue types listed at the top are described in “Materials and Methods.” Each reaction was performed in triplicate, and se calculations are reported in Supplemental Table S3. All values were normalized to Ubiquitin-Conjugating Enzyme (At5g25760), and relative levels are scaled to expression in seedlings. CYCB1;1 (At4g37490) was used as a marker for cell proliferation. The abundance of MCM2 transcripts in cultured cells was verified in multiple experiments. B, Immunoblot analysis of MCM5 and MCM7 protein abundance in equivalent tissues to those used in A. Total protein extract (50 μg) from cultured cells (lane 1), seedling (2 weeks; lane 2), shoot apex (lane 3), elongating leaf (<1 cm; lane 4), mature leaf (>2 cm; lane 5), senescing leaf (lane 6), cauline leaf (>2 cm; lane 7), cauline leaf (<1 cm; lane 8), flower bud (lane 9), open flower (lane 10), and silique (lane 11) was resolved by SDS-PAGE, and the blots were probed with polyclonal antibodies specific for Arabidopsis MCM5 (top) and MCM7 (middle) proteins. A nonspecific band that reacted with the secondary antibody was used as a loading control (bottom).

Figure 2.

Localization of endogenous MCM5 and MCM7 proteins in cultured plant cells. A to F, Immunoperoxidase staining demonstrated that MCM5 (A and D) and MCM7 (B and E) displayed localization patterns consistent with nuclear compartmentalization in both Arabidopsis and tobacco cultured cells. The discrete staining patterns for MCM5 and MCM7 were distinct from the diffuse background staining obtained using normal rabbit (NR) control serum (C and F). G to N, Immunofluorescence microscopy revealed that MCM5 (green in G and H) and MCM7 (green in K and L) colocalized with DAPI-stained DNA (blue) in most tobacco cells but not when condensed chromosomes were visible (arrows). Differential interference contrast (DIC) images are shown in J and N. The images are typical, and these patterns were observed in many cells over multiple experiments. Bars = 10 μm (A and B), 100 μm (C), and 15 μm (D–N).

Figure 3.

Localization of endogenous MCM5 and MCM7 proteins in N. benthamiana root tips. Confocal microscopy was used to visualize immunofluorescence-labeled MCM5 (green in A, C, and D), MCM7 (green in E, G, and H), and histone H1 (blue) in longitudinal sections of root tips (approximately 30 μm thick). MCM5 (C) and MCM7 (G) colocalized with DNA in most cells. When condensed chromosomes were visible, MCM5 (arrows in A–D) and MCM7 (arrows and asterisks in E–H) did not colocalize with DNA. At high magnification (D and H), nuclear and nonnuclear localization patterns are clearly distinct. Bars = 20 μm (A–C), 10 μm (D), 50 μm (E–G), and 10 μm (H).

Figure 4.

Arabidopsis MCM5 and MCM7 proteins are nuclear in G1, S, and G2/M cells. A, Nuclei were isolated from formaldehyde-cross-linked Arabidopsis suspension cells and sorted into G1, S, and G2/M populations based on DNA content by FACS. B, After cross-link reversal, proteins from 200,000 nuclei in G1 (lane 1), S (lane 2), and G2/M (lane 3) phases of the cell cycle were resolved by SDS-PAGE, and the blots were probed with anti-MCM5 (top) and anti-MCM7 (bottom) antibodies. C, Cytoplasmic contamination did not account for the presence of MCM5 and MCM7 in G2/M phase nuclei. Proteins from FACS-sorted G1 (lane 2) and G2/M (lane 4) nuclei and from whole cell extract (WCE; lanes 1 and 3) were resolved by SDS-PAGE. Blots were probed with anti-MCM5 (top) and anti-UGPase (bottom) antibodies.

Figure 5.

Chromatin binding of DNA replication initiation proteins in asynchronous Arabidopsis cultured cells. A, Scheme of the biochemical fractionation methods. B, An asynchronous culture of Arabidopsis cells was subjected to the fractionation described in A. Proteins (50 μg) from the whole cell extract (WCE) and volume equivalents from the non-chromatin-associated (S1) and chromatin-bound (P1) fractions were resolved by SDS-PAGE, and the blots were probed with the indicated antibodies. C, The purified chromatin fraction (P1) was incubated with or without 1 unit of DNase I for 30 min at 25°C and fractionated as in A. Volume equivalents from the soluble (S2) and pellet (P2) fractions were resolved by SDS-PAGE, and blots were probed with the indicated antibodies. D, DNA was extracted from samples processed as in C, resolved on a 1% agarose gel, and visualized with ethidium bromide staining. E, The purified chromatin fraction (P1) was treated with the indicated concentrations of NaCl for 10 min at 4°C and fractionated as in A. Volume equivalents from the soluble (S2) and pellet (P2) fractions were resolved by SDS-PAGE, and blots were probed with the indicated antibodies.