Truncation or Deletion Event Truncation or Deletion Event Addressing the role of the length of G1 phase of the cell cycle on genetic instability in S. cerevisiae Yi H Chen, Sriram Srikant, Marco Fumasoni, Andrew Murray FAS Center for Systems Biology, Department of Molecular and Cellular Biology, Harvard University Abstract Mutant Construction Mutant Phenotypes Discussion Mutants show the expected cell cycle profile and cell size phenotypes: sic1D and PGAL-CLN3 in galactose show a shorter G1 phase than WT, while PGAL-CLN3 in glucose show a significantly longer one (A). Repression of CLN3 results in larger cells while its over-expression results in smaller cells (B). G1 cell cycle regulators are often mutated in cancer cells, which which are characteristically genetically unstable. However, the mechanism by which mutations in G1 cell cycle regulators cause genetic instability remains unclear. Previous work has shown that mutants with deletions in SIC1, encoding for a CDK inhibitor that prevents precocious entrance to S phase, is associated with high genetic instability. We tested the hypothesis that sic1D confer genetic instability by shortening the G1 phase of the cell cycle. Mutants that alter the length of G1 phase through different mechanisms were constructed. The constructed mutants did not show comparable genetic instability to sic1D cells. Our preliminary results suggest that genetic instability in sic1D is not linearly dependent on the length of the G1 phase of the cell cycle, but may instead depend on the lack of a specific Sic1 mediated function. The high efficiency of homologous recombination reactions in S. cerevisiae combined with the availability of several genetic markers allow fast and targeted genome editing. Goal: Shorten the length of the G1 phase of the cell cycle and examine the effect on genetic instability cln3 Mutants CLN3 overexpressed CLN3 suppressed Native CLN3 locus CLN3 cassette CLN3 PGAL URA3 Inducible CLN3 Selection on -URA Plate Transformation Galactose Medium Glucose Medium Knock Out Mutants WHI5 or SIC1 Native locus deletion cassette KAN Selection on +G418 Plate whi5Δ or sic1Δ A) B) G1 S G2 M Shorten G1 phase by perturbing Cln3, Whi5, and Sic1 Cell Cycle Profile G1/G2 ratio A) WT sic1D whi5D CLN3 (GLU) CLN3 (GAL) * Cell Size Average Size (µm) * B) WT sic1D whi5D CLN3 (GLU) CLN3 (GAL) Findings: sic1D showed increased mutation rates compared to WT. However, all the other mutants (whi5D and PGAL-CLN3 in galactose) with shortened G1 phase analyzed did not. DNA damaging agent HU did not specifically affect the fitness of cells with a shorter G1 phase. Conclusion: Preliminary results suggest that genetic instability is not linearly dependent on length of the G1 phase However, the cell cycle profile of the mutants demonstrate that G1/G2 ratio of sic1D is lower than that of mutants whi5D and PGAL-CLN3 in galactose . Further experiments are needed to rule out the possibility that the amplitude by which whi5D and PGAL-CLN3 in galactose shortened the G1 phase was not sufficient to confer genetic instability Introduction A) DNA content of logarithmically growing cells was measured by FACS. G1 and G2 DNA content peaks were fitted with normal curves and ratio of the number of cells in each phase was estimated. Means and standard deviations of G1/G2 ratio are plotted. B) Average cell size was calculated with a lognormal distribution of the measured cells population sizes. Means and standard deviations of the average cell size are plotted. A) whi5 and sic1 KO were constructed by transforming WT cells with DNA cassettes containing a Kanamycin resistance marker and homologous sequences to the native loci of each gene (in red). The CLN3 mutants were constructed by transforming WT cells with a cassette containing the URA3 gene, GAL promoter, CLN3 and homology to the native CLN3 locus for targeted integration (in red). The Cell Cycle The series of events that lead to the duplication of an eukaryotic cell is referred to as the cell cycle and is composed of four main consecutive phases: Gap1 (G1), Synthesis (S), Gap2 (G2) and Mitosis (M). Genetic Instability: Methods Genetic Instability: Results Fluctuation Test Experimental Approach: Mutation Rate Assay G1 S G2 M Cell Growth Prepare for DNA replication Growth Prepare for division DNA replication Growth stops Orderly division of two daughter cells START A) ADE2 CAN1 can1 ADE2 Point Mutation Chromosome V ADE2 CAN1 can1 ade2 Truncation or Deletion Event sic1D mutation rates are significantly higher than all other strains. PGAL-CLN3 in glucose, PGAL-CLN3 in galactose, and whi5D do not show statistically different mutation rates than WT. WT sic1D whi5D CLN3 (GLU) CLN3 (GAL) Truncation/Deletion Point Mutation Figure adopted from Alberts B et al., 2002. Future Work Address the hypothesis that whi5D and PGAL-CLN3 in galactose did not sufficiently shorten the G1 phase by creating other mutants Double mutant with combinations whi5D, PGAL-CLN3 and Cln3-1 can be constructed to achieve a stronger effect on G1. (cln3-1 mutants express Cln3-1, which do not possess the C-terminal portion of Cln3 that is targeted for protein degradation) Construct sic1D PGAL-CLN3 double mutant to test if the repression of Cln3, by lengthening the G1 phase, is able to suppress the sic1D genetic instability Expression of Sic1 under different promoters that restricts its activity to specific cell cycle phases to identify the temporal origin of its genetic instability Cell Cycle Regulation: G1/S Transition B) WT Growth on ARG- CAN+ can1 Mutation CAN1 can1 ade2 No growth on ADE-: Truncation or Deletion Event can1 ADE2 Growth on ADE-: Point Mutation Culture for 48h ADE- Replica Plate ARG- CAN+ Plate can1 sic1D whi5D PGAL CLN3 in Galactose PGAL CLN3 in Glucose Succession of cell cycle phases is tightly regulated. Commitment to DNA replication is a particularly important event in the cell cycle (G1 to S phase transition) Many regulatory molecules affect the transition though different mechanisms, among those: Sic1: inhibits CDK activity that is required for initiation of replication Whi5: represses transcription factors required for expression of genes needed for DNA replication Cln3: eliminates Whi5 to allow for transcription of genes needed for DNA replication and S phase entry Luria-Delbruk fluctuation test was performed to calculate mutation rates. Ten samples of each strain were cultured and plated as described in methods. Ma-Sandri-Sarker Maximum likelihood Estimator (MSS-MLE) was used to calculate rates of truncation/deletion (shown in purple) and point mutation (shown in blue). 95% confidence interval are represented. Initiation of DNA replication G1 transcriptional program Cell cycle entry G1/G0  S Adopted from: Enserink, 2011. DNA Damage Sensitivity Assay A) ADE2 CAN1 system was used to detect mutation events. ADE2 was introduced to chromosome V, 10kB towards the telomere from the native CAN1 locus. No essential genes were present left of CAN1 so fragments of the chromosome can be lost without affecting cell viability. B) ADE2 CAN1 cells were cultured 48h in non-selective medium and plated on canavanine to select for can1 mutants. Surviving colonies were replica plated on –ADE medium to distinguish between point mutation (can1 ADE2) and truncation or deletion events (can1 ade2). WT sic1D whi5D YPD YPD + HU A) PGAL- CLN3 YP+ Glucose YP + Glucose HU B) YP + Galactose YP + Galactose HU C) HU has a slightly stronger effect on the sic1D fitness compared to WT, and whi5D cells. References Lengronne A., Schwob E., 2002. The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1). Mol. Cell DOI: 9: 1067/1078.  Enserink JM., 2011. Cell Cycle Regulation of DNA Replication in S. cerevisiae, DNA Replication-Current Advances. InTech, DOI: 10.5772/19055. Singh, A., & Xu, Y.-J. (2016). The Cell Killing Mechanisms of Hydroxyurea. Genes, 7(11), 99. DOI: 10.3390/7110099 Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. An Overview of the Cell Cycle. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26869/ Experimental Approach: DNA Damaging Agent, Hydroxyurea Hydroxyurea induces replication stress that results in genetic instability Normal Conditions Exposure to HU DNA Breakage A G T C Replication machinery dissociates Repression of CLN3 demonstrates higher sensitivity to HU compared to the overexpression of CLN3. S Phase Fork Stalling A C T G A G dNTPs available HU inhibits dNTP Production A G T C STOP Hypothesis: Does shortening G1 phase increase genetic instability by decreasing amount of time for preparation of DNA replication? Hydroxyurea (HU) is an inhibitor of enzyme ribonucleotide reductase, which catalyzes production of dNTPs. Exposure to HU limits the substrate available for DNA polymerase, resulting in blockage in the replication fork. Prolonged fork stalling leads to collapse of the replication machinery, resulting in generation of replication-dependent DNA breaks. (Singh, 2016) Spot assay with HU: Cells were cultured for 2 days until complete saturation. 1:10 serial dilutions were made in YP and spotted on appropriate plates containing galactose or glucose only, or in presence of 200mM HU.