Faculty Opinions recommendation of Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast.

AbstractAcross eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

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A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

Journal of Biological Chemistry ◽

10.1074/jbc.m116.772368 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3062-3073 ◽

Cited By ~ 3

Author(s):

Irina Bruck ◽

Nalini Dhingra ◽

Daniel L. Kaplan

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Budding Yeast ◽

S Phase ◽

Replication Initiation ◽

Initiation Factor ◽

Minichromosome Maintenance ◽

Initiation Factors ◽

Complex Protein ◽

Amplification Mechanism

The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2–7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2–7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein Mcm4 alone and also of the Mcm2–7 complex and the dsDNA-loaded Mcm2–7 complex. We further demonstrate that Dpb11 can directly recruit DDK to Mcm4. A DDK phosphomimetic mutant of Mcm4 bound Dpb11 with substantially higher affinity than wild-type Mcm4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2–7. Furthermore, dsDNA-loaded Mcm2–7 harboring the DDK phosphomimetic Mcm4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2–7. We isolated a mutant of Dpb11 that is specifically defective for binding to Mcm4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased Mcm4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and Mcm4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase.

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The limiting DNA replication initiation factors Sld2 and Sld3 influence origin efficiency independent of origin firing time

10.1101/384644 ◽

2018 ◽

Author(s):

Kelsey L. Lynch ◽

Elizabeth X. Kwan ◽

Gina M. Alvino ◽

Bonita J. Brewer ◽

M.K. Raghuraman

Keyword(s):

Rdna Locus ◽

S Phase ◽

Replication Initiation ◽

Yeast Genome ◽

Phase Duration ◽

Origin Firing ◽

Dna Replication Initiation ◽

Initiation Factors ◽

Origin Activation ◽

Time Of Origin

AbstractChromosome replication in Saccharomyces cerevisiae is initiated from roughly 300 origins that are regulated both by DNA sequence and by the limited abundance of four trans-acting initiation proteins (Sld2, Sld3, Dpb11 and Dbf4, collectively called “SSDD”). We set out to determine how the association of Sld2 or Sld3 at origins contributes to time of origin activation and/or origin efficiency using auxin-induced protein degradation to further decrease their abundance. Depleting cells of either factor slows growth rate, increases S-phase duration, and causes viability defects, without activating the S phase checkpoint. Chr XII is uniquely unstable with breakage occurring specifically within the rDNA locus. The efficiency of the rDNA origin is decreased while the onset of replication initiation is unchanged. We found that origin efficiency is reduced uniformly across the unique portions of the yeast genome. We conclude that the abundance of Sld2 and Sld3 contribute primarily to origin efficiency.

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

PLoS Genetics ◽

10.1371/journal.pgen.1008430 ◽

2019 ◽

Vol 15 (10) ◽

pp. e1008430 ◽

Cited By ~ 3

Author(s):

Kelsey L. Lynch ◽

Gina M. Alvino ◽

Elizabeth X. Kwan ◽

Bonita J. Brewer ◽

M. K. Raghuraman

Keyword(s):

Replication Initiation ◽

Origin Firing ◽

Initiation Factors ◽

Firing Efficiency

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Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

10.1101/2020.07.04.187286 ◽

2020 ◽

Cited By ~ 3

Author(s):

Y. Hu ◽

A. Tareen ◽

Y-J. Sheu ◽

W. T. Ireland ◽

C. Speck ◽

...

Keyword(s):

Dna Replication ◽

Gene Silencing ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Separate Analysis ◽

Genome Replication ◽

Sequence Specificity ◽

Sequence Motifs ◽

Origin Firing ◽

Α Helix

AbstractDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

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