Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle

Abstract

Eukaryotic replication origins are defined by the ORC-dependent loading of the Mcm2-7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 complexes exhibit little co-localization with ORC or replication foci and can function as dormant origins. We dissected the mechanisms regulating the assembly and distribution of the Mcm2-7 complex in the Drosophila genome. We found that in the absence of cyclin E/Cdk2 activity, there was a 10-fold decrease in chromatin-associated Mcm2-7 relative to the levels found at the G1/S transition. The minimal amounts of Mcm2-7 loaded in the absence of cyclin E/Cdk2 activity were strictly localized to ORC binding sites. In contrast, cyclin E/Cdk2 activity was required for maximal loading of Mcm2-7 and a dramatic genome-wide reorganization of the distribution of Mcm2-7 that is shaped by active transcription. Thus, increasing cyclin E/Cdk2 activity over the course of G1 is not only critical for Mcm2-7 loading, but also for the distribution of the Mcm2-7 helicase prior to S-phase entry.

Keywords: DNA replication; Mcm2‐7; cell cycle; chromatin.

Figure 1

Cyclin E/Cdk2 activity modulates Mcm2-7 loading during G1

1. FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.

2. Analysis of Mcm2-7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2-7 by Western blot. A non-specific band is indicated by an asterisk (*).

3. Quantification of the ratio of chromatin-bound Mcm2-7 relative to Orc2 (log₁₀ scale) for a minimum of 11 replicates (mean ± SD).

Figure 2

The full complement of Mcm2-7 is loaded during an unperturbed G1/S transition Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2-7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2-7, Orc2, and Dacapo.

Figure 3

The cyclin E/Cdk2 kinase-dependent phase of Mcm2-7 loading requires the canonical pre-RC assembly pathway

1. Schematic of the experiment.

2. FACS profiles of DNA content for each cell population assayed.

3. Western blot analysis for Orc2 and Mcm2-7 in the nuclear chromatin fraction and Orc2, Mcm2-7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.

Figure 4

Mcm2-7 loading is restricted to ORC binding sites in the absence of cyclin E

1. Genome-wide analysis of ORC localization by ChIP-chip. ORC enrichment from asynchronous cells is depicted for a 5-Mb section of chromosome 2L.

2. Genome-wide analysis of Mcm2-7 localization in early G1 by ChIP-chip. Mcm2-7 enrichment from cyclin E RNAi-depleted cells is depicted for a 5-Mb section of chromosome 2L.

3. Venn diagram depicting the overlap between ORC and Mcm2-7 peaks.

Figure 5

The Mcm2-7 chromatin distribution in G1 is dependent on cyclin E/Cdk2 activity and the transcription machinery

1. Genome-wide analysis of Mcm2-7 localization at the G1/S transition by ChIP-chip. Mcm2-7 enrichment from HU-arrested cells is depicted for a 5-Mb section of chromosome 2L. Inset: transcribed (green) and non-transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.

2. Bimodal distribution of Mcm2-7 enrichment over transcribed and non-transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non-transcribed (red) genes.

3. “Meta”-gene analysis of Mcm2-7 enrichment for different deciles of gene expression and their aggregated probe intensities.

4. Mcm2-7 is displaced from chromatin by DNA replication. Genome-wide analysis of Mcm2-7 localization in late S-phase (6 h post HU release) by ChIP-chip. Mcm2-7 enrichment is depicted for a 10-Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.

5. Box-plots representing late S-phase Mcm2-7 ChIP signal found within early (246) or late (167) replication domains.

Figure 6

Model of pre-RC assembly and redistribution of Mcm2-7 in G1 Mcm2-7 is loaded at ORC binding sites immediately after entry into G1. As G1 progresses, increasing cyclin E/Cdk2 activity promotes the loading of additional Mcm2-7 complexes resulting in the full complement of Mcm2-7 being loaded by the end of G1. All Mcm2-7 loading is dependent on Cdc6 and Cdt1. Prior to or coinciding with the entry into S-phase, the full complement of Mcm2-7 redistributes along the chromosomes and is displaced from transcribed genes.