Multiple functions for Drosophila Mcm10 suggested through analysis of two Mcm10 mutant alleles

Abstract

DNA replication and the correct packaging of DNA into different states of chromatin are both essential processes in all eukaryotic cells. High-fidelity replication of DNA is essential for the transmission of genetic material to cells. Likewise the maintenance of the epigenetic chromatin states is essential to the faithful reproduction of the transcriptional state of the cell. It is becoming more apparent that these two processes are linked through interactions between DNA replication proteins and chromatin-associated proteins. In addition, more proteins are being discovered that have dual roles in both DNA replication and the maintenance of epigenetic states. We present an analysis of two Drosophila mutants in the conserved DNA replication protein Mcm10. A hypomorphic mutant demonstrates that Mcm10 has a role in heterochromatic silencing and chromosome condensation, while the analysis of a novel C-terminal truncation allele of Mcm10 suggests that an interaction with Mcm2 is not required for chromosome condensation and heterochromatic silencing but is important for DNA replication.

Figure 1.—

Alignment of multiple Mcm10 proteins and schematic of Mcm10 mutant alleles. (A) Alignment of Mcm10 proteins showing conserved zinc-finger motifs (solid bars), highly conserved regions (bars with dark shading, 23–40% similarity for metazoans), and moderately conserved regions (bars with light shading, 10–22% similarity for metazoans). (B) Schematic of Mcm10 gene region on chromosome 2L with P-element insertion sites indicated for the two Mcm10 alleles used in this study.

Figure 2.—

RT–PCR measurement of relative Mcm10 transcript levels in the two Mcm10 alleles. (A) Visualization of transcript levels in the respective genotypes with rp49 loading control. (B) Bar graph of transcript levels as a ratio of rp49 control show that transcription of Mcm10 is significantly lower in the Mcm10^Scim19 background.

Figure 3.—

Schematic of Mcm10^d08029 truncation allele, shift in Mcm10 protein mobility, and reduction in pupae size. (A) The Mcm10^d08029 allele is predicted to cut off 85 aa from the C terminus of the protein and remove a conserved zinc-finger domain. (B) Western blots probed with α-Mcm10 of protein extracts from early embryos laid by females with the genotypes +/Mcm10^d08029 and Mcm10^d08029/Mcm10^Scim19, respectively. Left and right, Mcm10 protein mobility is altered in the Mcm10^d08029-containing background, consistent with prediction from P-element insertion location. Right, Western blot showing reduced levels of full-length Mcm10 protein in Mcm10^Scim19/Mcm10^d08029-derived embryo extracts as compared to the truncation protein. Western blot of Mcm10 protein from homozygous fly lines shows similar protein levels and mobility shifts compared to the first two panels. (C) Measurement of average pupae size in wild type, Mcm10^Scim19, and Mcm10^d08029 shows that Mcm10^d08029 pupae are on average 16% smaller than wild type (P < 0.00001).

Figure 4.—

Two-hybrid dissection of Mcm10 interaction domains. (A) Two-hybrid interactions indicated by growth on media lacking histidine between Mcm10 and Orc2, Mcm2, and Hp1, respectively. Growth was not observed on empty controls. (B) Schematic representation of Mcm10 fragments tested against the interactions in A. Centered directly below each fragment are results for growth on media lacking histidine with growth indicating a positive interaction. Note that fragment 1–100 aa of Mcm10 demonstrated one-hybrid activity. (C) Representation of the results in B showing regions of Mcm10 interaction with the proteins tested.

Figure 5.—

Serial dilution yeast two-hybrid testing of the effect of the Mcm10^d08029 allele on known protein interactions. The left column shows growth on media lacking histidine and the right column shows growth control on media with histidine. The top panel shows one-hybrid control showing no growth for empty vector controls. The middle panel shows the relative strength of two-hybrid interactions between Mcm10 and the proteins indicated. The bottom panel demonstrates the effect of the Mcm10^d08029 85-aa C-terminal truncation on the relative strength of these same interactions.

Figure 6.—

Effects of the two Mcm10 alleles on polytene chromosomes, DNA replication in larval brains, and mitotic index. (A) Confocal micrographs of polytene chromosome spreads from the genotypes indicated. Mcm10^d08029 polytene chromosomes are underreplicated compared to wt control and Mcm10^Scim19. (B) Fluorescent micrographs of wandering third instar larval brains showing DNA (blue) and EdU incorporation (green). wt brains are larger and show less EdU incorporation than either of the Mcm10 mutant alleles. (C) Graph of fraction of cells in mitosis for brain squashes of genotypes indicated.

Figure 7.—

Chromosomal phenotypes of the two Mcm10 alleles in larval brains and early embryos. (A) Fluorescent micrographs of representative mitotic figures from brain squashes of the indicated genotype. No significant differences were observed. (B) Fluorescent micrographs of nuclei in early embryos from the homozygous females for the two Mcm10 alleles and the wild-type control as well as the percentage of embryos showing two or more anaphase bridges per field of view. Significant cell-cycle asynchrony is observed in the Mcm10^Scim19 background as well as anaphase bridges (open arrows), while no anaphase bridges were observed in wt or Mcm10^d08029.

Figure 8.—

Confocal micrographs of Drosophila egg chambers and nurse cell nuclei. (A) String of egg chambers with different stages labeled. Note five-blob nurse cell nuclei at stage 5. By stage 7 all nuclei appear decondensed (some egg chambers have been reoriented for clarity). (B) Egg chambers at stages 5 and 7 from the two Mcm10 alleles and wt. The five-blob stage is present in all genotypes at stage 5. However, at stage 7 some nuclei in the Mcm10^Scim19 background have not decondensed and remain in the five-blob state (see inset).

Figure 9.—

PEV analysis of Mcm10 alleles and Hp1⁵ using a variegating dumpy reporter line. (A) Fraction of flies in each phenotypic class (0–5) for the genotypes indicated. Hp1⁵ results in a dramatic shift toward wild-type distribution of dumpy phenotypes. (B) Average “dumpy” score for the different genotypes. Both Hp1⁵ and Mcm10^Scim19 show significant suppression of dumpy PEV whereas Mcm10^d08029 shows no shift from wild type.