The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast

Lynch, Kelsey L.; Alvino, Gina M.; Kwan, Elizabeth; Brewer, Bonita J.; Raghuraman, M. K.

doi:10.1371/journal.pgen.1008430

Cited by 15 publications

(23 citation statements)

References 85 publications

(150 reference statements)

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“…These mutants would be forced to use late origins under HU to survive. This is also consistent with the observation that the peak widths on average for these slow growing mutants was larger than WT peak heights because the smaller number of origins utilized would not be restrained by rate-limiting replication factors, which are known to exist 16,17 . We suggest that the mutations at Orc4 R478 and N489 would not cause sequence specificity changes but instead cause inefficient binding to origins.…”

Section: Resultssupporting

confidence: 89%

Evolution of DNA replication origin specification and gene silencing mechanisms

Tareen

Sheu

et al. 2020

Nat Commun

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DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

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Section: Resultssupporting

confidence: 89%

Evolution of DNA replication origin specification and gene silencing mechanisms

Tareen

Sheu

et al. 2020

Nat Commun

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“…We propose that rDNA copy number reduction will affect genome replication only if the rDNA replication timing is affected. This proposal is supported by our earlier studies describing a mutant that induces replication stress and reduces rDNA copy number (250 to 90) (SLD3-AID, (Lynch et al, 2019)). The reduced rDNA array remained late replicating, possibly because its copy number reduction did not suffice to alter replication timing.…”

Section: Discussionsupporting

confidence: 67%

“…While ribosome deficiencies do result when rDNA copy number drops below a threshold (Delany et al, 1994;French et al, 2003;Ritossa and Atwood, 1966;Sanchez et al, 2017), several other phenotypes have been associated recently with more physiologically relevant rDNA copy number variants that are sufficient for ribosome biogenesis (Gibbons et al, 2014;Ide et al, 2010;Lu et al, 2018;Michel et al, 2005;Paredes et al, 2011;Picart-Picolo et al, 2020;Wang and Lemos, 2017;Xu et al, 2017). Additionally, the plastic nature of the rDNA locus allows copy number fluctuations in the face of stressors such as nutrient availability (Aldrich and Maggert, 2015) or replication defects (Ide et al, 2007;Lynch et al, 2019;Salim et al, 2017;Sanchez et al, 2017). We were intrigued by the rDNA reductions seen in the presence of replication stress, which strongly suggested that rDNA copy number variation might directly influence replication of the genome.…”

Section: Introductionmentioning

confidence: 99%

Coordination of genome replication and anaphase entry by rDNA copy number inS. cerevisiae

Kwan

Alvino

Lynch

et al. 2021

Preprint

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Ribosomal DNA (rDNA) copy number varies widely among individuals in many species, but the phenotypic consequences of such copy number fluctuations remain largely unexplored. In the yeast Saccharomyces cerevisiae, each rDNA repeat contains an origin of replication. Previous studies have demonstrated that the yeast rDNA locus can be a significant competitor for replication resources, suggesting that rDNA copy number variation may affect timely completion of genome-wide replication. We hypothesized that reduction in rDNA copy number and thus rDNA replication origins would reduce competition from the rDNA locus and thereby improve non-rDNA genome replication. To test this hypothesis, we engineered yeast strains with short rDNA arrays of 35 copies, a minimal copy number that still maintains wild type level ribosome function. Contrary to our hypothesis, the minimal rDNA strain displayed classic replication defects: decreased plasmid maintenance, delayed completion of chromosomal replication, and increased sensitivity to replication stress agonists. Although a normal rDNA array replicates late in S phase, the minimal rDNA array initiated replication in early S phase, resulting in delayed replication across the non-rDNA portions of the genome. Moreover, we discovered that absence of the rDNA fork barrier protein Fob1p increased DNA damage sensitivity in strains with early replicating rDNA. We present evidence that this increased sensitivity may be due to compromised regulation of cyclin phosphatase Cdc14p and premature entry into anaphase. Our results indicate that precocious rDNA replication, rather than total number of rDNA origins, compromises replication of the genome. Taken together, we suggest that the rDNA's large, late-replicating state is evolutionarily conserved to promote genome stability through timely genome replication and coordination of S phase completion with anaphase entry.

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“…Our previous work supports a model in which multiple MCMs are loaded at origins (Das et al, 2015;Yang et al, 2010). Furthermore, overexpression of origin-activating factors in S phase causes most all origins to fire early in S phase, consistent with most origins having at least one MCM loaded (Lynch et al, 2019). Our MNase approach to MCM ChIP-seq reported here further supports the multiple-MCM model by producing high-resolution maps of MCM locations that identify MCMs at multiple locations across origins.…”

Section: Discussionsupporting

confidence: 86%

Competition for MCM Loading at Origins Establishes Replication Timing Patterns

Dukaj

Rhind

2020

Preprint

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Loading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing.

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The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast

Cited by 15 publications

References 85 publications

Evolution of DNA replication origin specification and gene silencing mechanisms

Evolution of DNA replication origin specification and gene silencing mechanisms

Coordination of genome replication and anaphase entry by rDNA copy number inS. cerevisiae

Competition for MCM Loading at Origins Establishes Replication Timing Patterns

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