Proper initiation of chromosomal DNA replication requires the Sld3-Sld7 complex in budding yeast

　　Eukaryotic chromosomal DNA replication initiates from replication origins only once per cell cycle. Many proteins assemble on origins, form replication machinery, and then dissociate from origins to start DNA synthesis. However, the mechanism governing these processes has not been well elucidated. Here, l show that the Sld7 protein participates in the dissociation process between replication machinery and origins. SLD7 (Synthetic Lethality with Dpb 11-24　7), a newly identified gene in a synthetic lethal screening, is not essential for cell growth but required for several cellular processes, such　as an efficient chromosomal DNA replication. The Sld7 protein always forms a complex with the Sld3 protein, which functions for the initiation of chromosomal DNA replication, to enhance the Sld3 function. Thus, high-copy SLD3 gene compensates for the lack of Sld7, and high-copy SLD7 gene suppresses the sld3-6 mutation that reduces the complex　formation between Sld3 and Sld7 proteins. This Sld3-Sld7 complex associates with replication origins in Gl -phase and dissociates gradualty from them when replication initiates. Sld3 protein associates with and then dissociates from origins in the absence of SId7. The Mcm7 protein, a component of replication machinery, associates with origins and then with neighboring regions of origins as it moves with replication forks. In the absence of Sld7 protein, although Mcm7 associates with origins as efficient as in WT cells, its association with neighboring region is significantly reduced. Therefore, the Sld7 protein seems to help the efficient displacement of replication machinery from origins. <br /

Proper initiation of chromosomal DNA replication requires the Sld3-Sld7 complex in budding yeast

　　Eukaryotic chromosomal DNA replication initiates from replication origins only once per cell cycle. Many proteins assemble on origins, form replication machinery, and then dissociate from origins to start DNA synthesis. However, the mechanism governing these processes has not been well elucidated. Here, l show that the Sld7 protein participates in the dissociation process between replication machinery and origins. SLD7 (Synthetic Lethality with Dpb 11-24　7), a newly identified gene in a synthetic lethal screening, is not essential for cell growth but required for several cellular processes, such　as an efficient chromosomal DNA replication. The Sld7 protein always forms a complex with the Sld3 protein, which functions for the initiation of chromosomal DNA replication, to enhance the Sld3 function. Thus, high-copy SLD3 gene compensates for the lack of Sld7, and high-copy SLD7 gene suppresses the sld3-6 mutation that reduces the complex　formation between Sld3 and Sld7 proteins. This Sld3-Sld7 complex associates with replication origins in Gl -phase and dissociates gradualty from them when replication initiates. Sld3 protein associates with and then dissociates from origins in the absence of SId7. The Mcm7 protein, a component of replication machinery, associates with origins and then with neighboring regions of origins as it moves with replication forks. In the absence of Sld7 protein, although Mcm7 associates with origins as efficient as in WT cells, its association with neighboring region is significantly reduced. Therefore, the Sld7 protein seems to help the efficient displacement of replication machinery from origins. <br /

Biological Monitoring of human exposure to neonicotinoids using urine samples, and neonicotinoid excretion kinetics

[Background] Neonicotinoids, which are novel pesticides, have entered into usage around the world because they are selectively toxic to arthropods and relatively non-Toxic to vertebrates. It has been suggested that several neonicotinoids cause neurodevelopmental toxicity in mammals. The aim was to establish the relationship between oral intake and urinary excretion of neonicotinoids by humans to facilitate biological monitoring, and to estimate dietary neonicotinoid intakes by Japanese adults. [Methodology/Principal Findings] Deuterium-labeled neonicotinoid (acetamiprid, clothianidin, dinotefuran, and imidacloprid) microdoses were orally ingested by nine healthy adults, and 24 h pooled urine samples were collected for 4 consecutive days after dosing. The excretion kinetics were modeled using one- and two-compartment models, then validated in a non-deuterium-labeled neonicotinoid microdose study involving 12 healthy adults. Increased urinary concentrations of labeled neonicotinoids were observed after dosing. Clothianidin was recovered unchanged within 3 days, and most dinotefuran was recovered unchanged within 1 day. Around 10% of the imidacloprid dose was excreted unchanged. Most of the acetamiprid was metabolized to desmethyl-Acetamiprid. Spot urine samples from 373 Japanese adults were analyzed for neonicotinoids, and daily intakes were estimated. The estimated average daily intake of these neonicotinoids was 0.53-3.66 μg/day. The highest intake of any of the neonicotinoids in the study population was 64.5 μg/day for dinotefuran, and this was <1% of the acceptable daily intake