The absence of F-box motif Encoding Gene SAF1 and Chromatin Associated factor CTF8 together contributes to MMS Resistant and HU Sensitive phenotype in S.cerevisiae

Abstract

11The Replication factor-C compex which related to cohesion, constitutes, three subunits called 12 Ctf18, Ctf8 and Dcc1. These three subunit complex assist the loading of PCNA onto the 13 chromosome. None of the replication factor C components are essential for cell viability. The 14 null mutant of the CTF8 in S.cerevisiae shows the chromosome instability and high frequency of 15 chromosome loss. The SAF1 gene product of S. cerevisiae involved in the degradation of 16 adenine deaminase factor Aah1p by SCF-E3 ligase, which itself is the part of E3 ligase. The 17 ubiquitin marked degradation of Aah1p occurs during nutrient stress which lead to cell enter into 18 the quiescent state. The N-terminus of Saf1p interacts with the Skp1 of SCF-E3 ligase and at C-19 terminus recruits with Aah1p. Here we have investigated about the binary genetic interaction 20 2 between the SAF1 and CTF8 genes. The strains containing single and double gene deletions of 21 SAF1 and CTF8 were constructed in the BY4741 genetic background. Further the mutant strains 22 were evaluated for growth fitness, genome stability and response to genotoxic stress caused 23 by hydroxyurea (HU) and methyl methane sulfonate (MMS). The saf1Δctf8Δ strain showed the 24 increased growth phenotype in comparison to saf1Δ, ctf8Δ, and WT strain on YPD medium. 25However saf1Δctf8Δ strain when grown in the presence MMS showed resistance and HU 26 sensitive phenotype when compared with saf1Δ, ctf8Δ. The frequency of Ty1 retro-transposition 27 was also elevated in saf1Δctf8Δ in comparison to either saf1Δ or ctf8Δ. The number of cells 28 showing the two or multi-nuclei phenotype was also increased in saf1Δctf8Δ cells when 29 compared with the either saf1Δ or ctf8Δ. Based on these observations, we report that the absence 30 of both the gene SAF1 and CTF8 together leads to MMS resistance, HU sensitivity, and genome 31 instability. This report warrants the investigation of mechanisms of differential growth 32 phenotype due to loss of SAF1 and CTF8 together in presence of genotoxic stress in future. 33 Introduction: 34 The ubiquitin proteasome system regulates the phase transition due to nutrient availability in 35 Saccharomyces cerevisiae (FINLEY et al. 1987).The nutrient deprivation condition induces stress, 36 which leads to cell enter into the quiescent phase (FINLEY et al. 1987).The Saf1, of S.cerevisiae 37 which constitutes the SCF E3-ligase component, recruits the Aah1p, for proteasomal mediated 38 degradation during nutrient deprivation conditions (ESCUSA et al. 2006; ESCUSA et al. 2007). The 39 Aah1, adenine deaminase of S.cerevisiae, converts adenine to hypoxanthine, implicating the role 40 of Saf1 in nucleotide metabolism. The genetic interaction studies reported negative interaction 41 of SAF1 with CDC10, CDC11, CDC12, and HYP2 (COSTANZO et al. 2016). The null mutant of 42 SAF1 showed the synthetic growth defects with HSP82 (ZHAO et al. 2005), POL2 (DUBARRY et HIS3 is split by artificial intron (AI) in an orientation opposite to the HIS3. When Ty1 which is 12...

Genetic interaction between glyoxylate pathway regulator UCC1 and La‐motif‐encoding SRO9 regulates stress response and growth rate improvement in Saccharomyces cerevisiae

“…For growth, a single colony of each strain was streaked on YPD + agar plates and incubated at 30°C for 2-3 days [26,27]. For growth kinetics, cultures of each strain were grown in liquid media (YPD broth) in triplicates, at 30°C with 200-rpm shaking.…”

“…Semiquantitative spot assay [26] was performed for cellular growth response of each strain in the presence of alternate carbon source and stress agents on the YPD solid media. Yeast cells were grown in YPD liquid medium at 30°C, with 200-rpm shaking until log phase was attained.…”

“…Cells from each strain were grown until log phase was attained to elucidate morphology by phase-contrast microscopy [26], using Leica DM3000 microscope. The process was followed by calcofluor white (CFW) staining [26,28,29].…”

“…Cells from each strain were grown until log phase was attained to elucidate morphology by phase-contrast microscopy [26], using Leica DM3000 microscope. The process was followed by calcofluor white (CFW) staining [26,28,29]. Cells collected by centrifugation were washed twice with 1× phosphate buffer saline and stained with CFW fluorescent dye (50 µg/ml).…”

“…The list of strains used for the study is provided in Table 1. Deletion of ORFs was conducted as per protocols [25,26]. For ORF deletion, a selectable marker cassette with 40 bp homology to either end of the target ORF was a polymerase chain reaction generated from plasmids (Table 2), using Kapa Taq Polymerase (Sigma-Aldrich).…”