Anti-Sml1 | Suppressor of Mec1 lethality

Sml1is a ribonucleotide reductase inhibitor involved in regulation of dNTP production. It is regulated by Mec1p and Rad53p during DNA damage and S phase. Synonymes: Yml058w.

Cell preparation for western blot: cells were harvested by centrifugation (4000 x g , 6 min, 4°C). Supernatant was discarded and cells were resuspended in 500 μl cold TCA buffer (20 mM Tris, pH 8, 50 mM ammonium acetate, 2 mM EDTA, 1 tablet/10 ml of Complete Mini Protease inhibitor cocktail with EDTA (Roche Diagnostics GmbH)). 500 μl 0.5 mm Zirconia/Silica Beads (BioSpec Products, Inc, 11079105z) and 500 μl cold 20% TCA was added. Samples were vigorously vortexed twice for 30 s (kept on ice in between), 750 μl from the liquid phase was transferred into a fresh Eppendorf tube. Samples were centrifuged for 10 min (20000 x g, 4°C). The pellet was resuspended in 300 μl TCA-Laemmli buffer and boiled for 10 min at 100°C.

Cerritelliet al. (2020). High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase. Nucleic Acids ResCorcoles-Saezet al. (2019). Essential Function of Mec1, the Budding Yeast ATM/ATR Checkpoint-Response Kinase, in Protein Homeostasis. Dev Cell. 2018 Aug 20;46(4):495-503.e2. doi: 10.1016/j.devcel.2018.07.011.Garbaczet al. (2019). The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity. Nucleic Acids Res. 2019 Jan 30. doi: 10.1093/nar/gkz048.Gollaet al. (2017). A systematic assessment of chemical, genetic, and epigenetic factors influencing the activity of anticancer drug KP1019 (FFC14A). Oncotarget. 2017 Sep 30;8(58):98426-98454. doi: 10.18632/oncotarget.21416.Dmowskiet al. (2017). Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint. PLoS Genet. 2017 Jan 20;13(1):e1006572. doi: 10.1371/journal.pgen.1006572.Mertzet al. (2015). Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity. Proc Natl Acad Sci U S A. 2015 May 12;112(19):E2467-76. doi: 10.1073/pnas.1422934112. Epub 2015 Mar 31.Singhet al. (2014). Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae. FEBS Lett. 2014 Feb 20. pii: S0014-5793(14)00137-9. doi: 10.1016/j.febslet.2014.02.017.Azadet al. (2013). Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in Saccharomyces cerevisiae. PLoS One, March 8.Poliet al. (2012).dNTP pools determine fork progression and origin usage under replication stress. The EMBO J. January 2012, 1-12.

Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.