No Origin, No Problem for Yeast DNA Replication

Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism

Non-canonical origins arise around deleted ARS sites.

<p>BrdU IP-ChIP replication profiles of chromosome VI. Wild-type (blue) and <i>7ori</i>Δ (green) cells were arrested in G1 and released into media containing BrdU and 200 mM HU for 60 min. The Y-axes show BrdU signal ratio in log₂ scale using a non-replicated sample (G1) as a reference, and the X-axes show chromosomal coordinates in kilobases. Deleted ARSs are labeled with grey arrows and ARSs that remained intact are labeled with black arrows. The red bar below <i>7ori</i>Δ, near left telomere, denotes the region that has high homology to other chromosomes. Therefore, the elevated BrdU signal at this region is likely due to cross-hybridization with DNA fragments corresponding to other chromosomal loci.</p

Strains used in this study.

<p>Strains used in this study.</p

<i>7ori</i>Δ origin deletions.

<p><i>7ori</i>Δ origin deletions.</p

Deleted origins display Orc2 depletion and maintain robust BrdU incorporation.

<p>Alignment of Orc2 enrichment and BrdU incorporation in wild-type (blue) and <i>7ori</i>Δ (green) cells at (a) <i>ARS604</i> (<i>ars604::loxP in 7ori</i>Δ) and (b) <i>ARS605</i> (<i>ars605::loxP</i> in <i>7ori</i>Δ). Black bar denotes ARS sequences.</p

Chromosome VI <i>7ori</i>Δ ARS deletion strain does not exhibit gross growth defects even in the presence of replication stress.

<p>(a) A schematic drawing of ARS locations on chromosome VI. ARS sequence positions are represented by a black bar and labeled with an arrow. The centromere is denoted by a black circle. Deleted ARSs on the <i>7ori</i>Δ chromosome VI are labeled with grey arrows and ARSs that remained intact are labeled with black arrows. (b) Five-fold serial dilution assay of wild-type and 7<i>ori</i>Δ on indicated media. The <i>mec1</i>Δ<i> sml1</i>Δ S-phase checkpoint mutant is shown as a replication inhibitor positive control.</p

Deleted origins do not recruit Orc2.

<p>Orc2-3XFlag chromatin ChIP-Chip of (a) chromosome VI and (b) chromosome III (control) in G2/M arrested cells. Wild-type (blue) and <i>7ori</i>Δ (green). ARS positions are shown as black bars above each panel. The centromere is denoted by a black circle. Orc2 ChIP signals are shown, along with the difference between the wild-type and <i>7ori</i>Δ signal (black) to show level of Orc2 depletion in <i>7ori</i>Δ. The Y-axes show Orc2 signal ratio in log₂ scale, and the X-axes show chromosomal coordinates.</p

SCREAM/ICE1 and SCREAM2 Specify Three Cell-State Transitional Steps Leading to Arabidopsis Stomatal Differentiation[W][OA]

Differentiation of specialized cell types in multicellular organisms requires orchestrated actions of cell fate determinants. Stomata, valves on the plant epidermis, are formed through a series of differentiation events mediated by three closely related basic-helix-loop-helix proteins: SPEECHLESS (SPCH), MUTE, and FAMA. However, it is not known what mechanism coordinates their actions. Here, we identify two paralogous proteins, SCREAM (SCRM) and SCRM2, which directly interact with and specify the sequential actions of SPCH, MUTE, and FAMA. The gain-of-function mutation in SCRM exhibited constitutive stomatal differentiation in the epidermis. Conversely, successive loss of SCRM and SCRM2 recapitulated the phenotypes of fama, mute, and spch, indicating that SCRM and SCRM2 together determined successive initiation, proliferation, and terminal differentiation of stomatal cell lineages. Our findings identify the core regulatory units of stomatal differentiation and suggest a model strikingly similar to cell-type differentiation in animals. Surprisingly, map-based cloning revealed that SCRM is INDUCER OF CBF EXPRESSION1, a master regulator of freezing tolerance, thus implicating a potential link between the transcriptional regulation of environmental adaptation and development in plants

Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8)

Patterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development