Screening

suppressor mutations to identify mutations in the Mediator of

replication checkpoint protein 1 ( MRC1) gene in Saccharomyces cerevisiae
Elizabeth Daugherty , Pat Visanpattanasin , Ryan Masi , Javi
1 1 2 Casado , Carolyn R. Norris
1 1
Departments of Biology1, Chemistry2, Johns Hopkins University, Baltimore, Maryland, USA
Contact: crn@jhu.edu

INTRODUCTION RESULTS & FIGURES CONCLUSIONS

Endocytosis is a process that allows proteins and other cargo to • A mutation in MRC1 allows for growth at 38 ℃ and rescues
travel across the plasma membrane of cells. In Saccharomyces endocytic activity in S. cerevisiae by an unknown pathway
cerevisiae, only wild-type cells or cells with intact ENT domain are • MRC1 is an S-phase checkpoint protein that is necessary for
viable at 38 ℃. Cells that have a quadruple deletion of endocytic DNA replication. A mutation in MRC1 increases duration of S-
adaptors (ΔΔΔΔ+ENTH) are not viable at 38 ℃ and exhibit impaired phase, which leads to increased DNA replication and results in
endocytosis. A spontaneous mutation caused by errors in DNA increased chromosomal instability that allows for more
replication can occur that suppresses the mutant phenotype and mutation events to arise in genes
reverts the phenotype back to wild-type. In our experiment, we • High mutation rate caused by a mutation in MRC1 might lead to
screened for a suppressor mutation in various S. cerevisiae BWY Figure 1A: Ptr2-GFP localization at the Figure 1B: Ptr2-GFP localization at the a mutation in genes that are involved in endocytosis.
strains (6959, 6960, 7111, 7112) that would allow the ΔΔΔΔ+ENTH to plasma membrane in ∆∆∆∆+ENTH vacuoles in ∆∆∆∆+ENTH mutant strain, • Endocytosis is rescued indirectly by a mutation in MRC1
grow at 38 ℃ and restore the endocytic activity. Several mutants parent strain where endocytic activity is observed to
were selected and categorized into complementation groups have been restored
depending on their phenotypes. Promising mutants were finalized FUTURE EXPERIMENTS
and sent for sequencing. The result was aligned with the reference
and parent sequences before the mutated gene was identified. • Since so little is known about how MRC1 actually affects
We have found that MRC1, located between positions 18816 and endocytosis in S. cerevisiae, it would be interesting to
22106 on chromosome III, affects the growth of cells at 38 ℃ and investigate the role of MRC1 in endocytosis
restores endocytic activity. MRC1 is an S-phase checkpoint protein • Comparisons between MRC1 functions in S. cerevisiae and its
that is necessary for DNA replication; however, it is yet unclear homolog, Claspin4, in humans would be interesting, with
whether this gene directly affects endocytosis, or the endocytosis is further possible applications in medicine and scientific
Figure 2: Location of the mutation on MRC1 (red arrow) and the MRC1 location on
affected due to the MRC1 interfering with genes that are involved discovery
Chromosome III (18816-22106)
with endocytosis.
• Comparison to parent and reference (saccer3): There are approximately 5-10 LITERATURE CITED
MATERIALS & METHODS differences between both the mutant and the parent as well as the mutant and
1. Alcasabas AA, Osborn AJ, Bachant J, Hu F, Werler PJH, Bousset K,
the reference, whereas the differences between each number in the thousands
Furuya K, Diffley JFX, Carr AM, Elledge Stephen J. 2001. Mrc1
Cross each mating type
• Identification of mutation: MRC1 is a protein coding gene, and was identified transduces signals of DNA replication stress to activate Rad53.
with parent strain on
Viable MATa at 38 ℃ on using BowTie2 and Free Bayes from Whole Genome Alignment (WGA), and Nat. Cell Biol 3:958–965.
YPD
Cross MATa and MATα YPD (mutant ΔΔΔΔ+ENTH) mutations were subsequently isolated using various Excel functions 2. Huiqiang Lou H, Komata M, Katou Yuki, Guan Z, Reis CC, Budd M,
at 30 ℃ on YPD Shirahige K, Campbell Judith L. 2008. Mrc1 and DNA Polymerase
• Isolation of mutation: From S. cerevisiae colonies that were grown on YPD ɛ Function Together in Linking DNA Replication and the S Phase
Viable MATα at 38
℃ on YPD plates, a select number of mutants were chosen to undergo DNA isolation Checkpoint. Mol. Cell 32:106–117.
(mutant Nonviable at 38 ℃ on YPD 3. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T,
Grow diploids at 30 ΔΔΔΔ+ENTH) (recessive mutation) or viable 38 Figure 3A: Right- Figure 3B: Sugimoto K, Shirahige K. 2003. S-phase checkpoint proteins Tof1
℃ on YNB-TRP- ℃ on YPD (dominant mutation) side up view of and Mrc1 form a stable replication-pausing complex. Nature
Upside down
URA Replica plate at 30 ∆∆∆∆+ENTH view of 424:1078–1083.
℃ from YPD to mutant 13 at 38 ∆∆∆∆+ENTH 4. Lin SY, Li K, Stewart GS, Elledge SJ. 2004. Human Claspin works
Viable at 30 ℃ on YNB-TRP-URA ℃ on YPD plates mutant 13 at 38 with BRCA1 to both positively and negatively regulate cell
Nonviable at 30 ℃
YNB-TRP-TURA (select for ℃ on YPD plates proliferation. PNAS 101(17):6484–6489.
on YNB (haploids)
(haploids) diploids) 5. MRC1 / YCL061C Overview. Saccharomyces Genome Database;
Replica plate at 30 ℃ [accessed 2017 Dec 3]. https://www.yeastgenome.org/locus/S0
00000566
Viable at 38 ℃ on
from YNB-TRP-URA to
Grow at 38 ℃ YPD (grow diploids)
FUNCTION OF MRC1 6. Osborn AJ, Elledge SJ. 2003. Mrc1 is a replication fork component
YPD (same
complementation or 30 ℃ on YPD • MRC1 is known to be involved with a number of various processes related to DNA whose phosphorylation in response to DNA replication stress
group) or nonviable replication, such as coupling together DNA helicase and polymerase and activates Rad53. Genes Dev 17:1755–1767.
at 38 ℃ on YPD stabilizing certain subunits of DNA polymerases that can be found at stalled
(different Isolation of DNA using API buffer,
replication forks when the cell is experiencing stress
complementation RNAase A, buffer P3, buffer AW2, TE
• It is also defines a novel S-phase checkpoint that coordinates both the replication
ACKNOWLEDGEMENTS
group) buffer
and transcription of DNA during “osmostress” We would like to thank Dr. Carolyn Norris, Katarzyna (Kasia) Hussey,
• MRC1 also protects uncapped telomeres, thereby ensuring the safety of strands and Neta Schwartz for their wealth of knowledge regarding
Identify and of DNA experimental design and data analysis. Special thanks to our
Compare mutant characterize • Claspin, which is a homolog of MRC1 in humans, is required for resistance to parents and other strong supporters (Viruch Visanpattanasin &
Determine
Assemble sequence to reference genes using multiple forms of genotoxic stress including UV and IR4. Another function of
number of unique Supaluck Suwanaroon, Kenneth Daugherty & Barbara Daugherty,
sequenced (saccer3) and parent Saccharomyces Claspin is that it activates the tumor suppressor Chk1 in response to damage to
mutations using Christopher Masi & Grace Masi, and Grace Lee ) for their
data using sequences using Genome DNA
Excel encouragement in all that we do.
BowTie2 FreeBayes Database