Disruption of mechanisms that prevent rereplication triggers a DNA damage response

Abstract

Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6(Delta)NT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6(Delta)NT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.

FIG. 1.

Induction of rereplication. (A) Mutations in ORC6, ORC2, MCM7, and CDC6 all contribute to increasing the extent of rereplication, as revealed by FACS. Cells with the indicated genotypes were cultured in YPR. Galactose (3%) was added, and the cells were incubated for an additional 4 h. (B) Induction of GAL-CDC6ΔNT-HAm. Cells from part A were probed for Cdc6ΔNT-HA by Western blotting against hemagglutinin (HA). Amido-black staining of the blot is shown as a loading control. (C) Cells from panel A were subjected to FACS analysis at a different scale (lower voltage) to expose the complete range of DNA contents. (D) Rereplicating cells arrest as large buds with a short spindle. ORC6-ps,rxl ORC2-ps MCM7-NLS GAL-CDC6ΔNT-HAm TUB1-GFP cells were treated as described for part A. Differential interference contrast (DIC) and fluorescence pictures were taken.

FIG. 2.

Genes found to interact with mutations compromising rereplication controls (ORC6-rxl GAL-CDC6ΔNT-HAs) are known to interact in a network. Clusters of genes that caused synthetic lethality or rescued ORC6-rxl GAL-CDC6ΔNT-HAs strains (Table 1) were mapped with Osprey software, using information from the GRID public database (the figure is adapted from the Osprey display). Line colors refer to the nature of the interaction (green, genetic interaction; pink, affinity purification; red, purified complex; blue, yeast two-hybrid interaction). Gene name colors refer to functional categories (red, DNA damage and replication stress response; blue, cell cycle; black, other functions). In our screen, genetic interactions were observed between all shown genes and ORC6-rxl CDC6ΔNT, but this information was omitted for clarity (a green line would link ORC6 CDC6 [at the center] to all genes). A pie chart is provided to show what fraction of genes recovered fell into each of the three main categories.

FIG. 3.

The survival of ORC6-rxl GAL-CDC6ΔNT-HAs cells depends on genes required for double-strand-break signaling and repair by homologous recombination. (A to F) Effects of introductions of gene deletions in the ORC6-rxl GAL-CDC6ΔNT-HAs background. Strains with the indicated genotypes were serially diluted 10-fold and plated on YEPD or YEP-Gal plates, and plates were incubated at 30°C for 2 days.

FIG. 4.

Strains with compromised rereplication controls accumulate Ddc2-GFP foci. (A) Cells were grown in YPD and transferred to YPR for 8 h, and then galactose (3%) was added. Cells were visualized after 3 h for Ddc2-GFP foci by DeltaVision microscopy. In addition to the indicated genotypes, all strains expressed DDC2-GFP. (B) Percentages of cells showing Ddc2-GFP foci in the presence of glucose (D; GAL-CDC6ΔNT-HA off) or galactose (G; GAL-CDC6ΔNT-HA on [grown as described for panel A]). Numbers describing strains correspond to the numbers in panel A. The data shown in panels A and B were obtained from the same experiment, with at least 100 cells scored for each condition.

FIG. 5.

Deletion of MRC1, TOF1, or CSM3 rescues ORC6-rxl CDC6ΔNT cells. Diploid strains with the following genotypes were sporulated, and tetrads were dissected: ORC6-rxl::LEU2/ORC6-wt CDC6ΔNT/CDC6-wt mrc1Δ::HIS3/MRC1-wt (A), ORC6-rxl::LEU2/ORC6-wt CDC6ΔNT/CDC6-wt tof11Δ::KanMX/TOF1-wt (B) and ORC6-rxl::LEU2/ORC6-wt CDC6ΔNT/CDC6-wt csm3Δ::KanMX//CSM3-wt (C). Spores were scored for growth without leucine, without histidine, and with G418. The presence or absence of the CDC6 N-terminal coding sequence was scored by PCR for all Leu⁺ spores for panel A and for all spores for panels B and C. In all crosses, all possible genotypes were recovered (data not shown). A strong slow-growth phenotype was noted for ORC6-rxl CDC6ΔNT (MRC1-wt, TOF1-wt, or CSM3-wt) spores (triangles) but not for ORC6-rxl CDC6ΔNT (mrc1Δ, tof11Δ, or csm3Δ) spores (squares).

FIG. 6.

ORC6-ps,rxl GAL-CDC6ΔNT-HAs cells activate Rad53 and accumulate replicated DNA in a MEC1-, RAD17-, DDC1-, and MRE11-dependent, but RAD52-independent, manner. Cells with the indicated genotypes were treated as described in the legend to Fig. 1A. Flag-Rad53 was probed for anti-Flag Western blotting. Maximal Flag-Rad53 activation was achieved by incubating cells with 0.1% MMS for 4 h. (A) Responses of GAL-CDC6ΔNT-HAs strains containing ORC6 (-wt, -ps, -rxl, or -ps,rxl) alleles. (B) Responses of ORC6-ps,rxl GAL-CDC6ΔNT-HAs strains containing different gene deletions.

FIG. 7.

Deletion of MEC1, MRE11, and RAD17, but not RAD52 or MRC1, allows more extensive rereplication. (A) Cells with the indicated genotypes were grown in YPR. Galactose (3%) was added, and the cells were incubated for an additional 4 h. (B) Induction of GAL-CDC6ΔNT-HAm. Cells from part A were probed for Cdc6ΔNT-HA by Western blotting against HA. Amido-black staining of the blot is shown as a loading control.

FIG. 8.

Pathways responding to loss of rereplication control. Previous studies established that Orc2 and Orc6 phosphorylation, nuclear exclusion of MCMs, Cdc6 proteolysis, and Orc6-Clb5 binding act to prevent pre-RC reassembly following replication initiation (see the introduction). In the absence of some of these controls, aberrant pre-RC assembly (and possibly DNA unwinding) can lead to DNA damage and even to extensive DNA rereplication that can cause more damage. The damaged DNA recruits the Mre11-Xrs2-Rad50 complex, the Mec1-Ddc2 complex, and the Rad17-Ddc1-Mec3 complex. These are required to signal the damage to Rad53, possibly allowing a premitotic cell cycle delay for DNA repair. These complexes also function in limiting the extent of rereplication. The DNA damage is repaired by homologous recombination dependent on Rad52 and possibly Srs2, Mms1, and Mms22. If and when the damage is completely repaired, the cell can resume proliferation. Other genes not diagrammed here, some of which were recovered in our genetic screen (like CTF4), could function in these or other pathways in response to rereplication.