Identifying Antimutators in Saccharomyces cerevisiae

Bekah Ashpole, Julia Joo, Stuart MacGeorge, Anna Lee, Brady Hearn, Kaelah Gendron, and Alan J. Herr
Department of Pathology, University of Washington
Abstract Key Questions Results
Mutator phenotypes due to mutations in genes encoding DNA polymerases or mismatch
repair proteins lead to increased error rates during DNA replication that accelerate the How do cells escape error-induced extinction? Extragenic antimutators affect genes involved in dNTP
evolution of cancer cells and contribute to chemotherapy resistance. Work in the yeast
Saccharomyces cerevisae indicates that excessive DNA replication errors can lead to
synthesis.
error-induced extinction (EEX), where every cell within the population dies due to a How do the affected pathways influence mutator-driven
random lethal mutation. Thus, a possible direction for cancer therapy may be to modulate cancers?
mutation rates of mutator cells – to either increase mutation rates to a lethal level or
suppress mutation rates to their baseline levels in order to slow the rate of tumor
evolution. Genetic pathways that may be targeted to modulate mutator phenotypes have
yet to be fully elucidated. To identify such pathways, we isolated spontaneous haploid
Methods
yeast eex mutants that escaped from error-induced extinction caused by combining a
mismatch repair defect (msh2Δ) with the pol2-L439V allele, which models a human Using an engineered yeast strain deficient in MMR and
cancer mutation affecting DNA polymerase epsilon. We crossed the eex mutants to wild
type yeast, generating diploid strains that were heterozygous for msh2Δ, pol2-L439V, and
the Pol ε exonuclease, we selected strains that escaped
the eex mutation. Following sporulation, we measured the mutation rates of haploid error-induced extinction.
MMR-proficient pol2-L439V progeny in order to assess the presence or absence of the
antimutator mutation. For each mapping experiment, cells showing evidence of
suppressed mutation rates were pooled separately from those exhibiting the normal
pol2-L439V mutator phenotype. Unique mutations present only in the antimutator pool
were identified by Illumina Next Generation Sequencing. Candidate eex mutations were
engineered into fresh strains to assess the antimutator phenotype. Our compiled list of
antimutator mutations and their functions in yeast may allow us to identify analogous
mutations in the human genome and explore new methods of cancer therapy by which
specific genes can be targeted to lower mutation rates to normal levels.
Antimutator mutations affecting Dun1, RNR1, and RNR2 may increase
replication fidelity by lowering dNTP pools. Ctf18 deficiency may also
Introduction affect dNTP pools through the S-phase checkpoint or directly influence
replication fidelity through the loss of its known association with Pol ε.
Polymerase proofreading and MMR cooperatively guard
against mutations.
Dissected colonies were genotyped and mutation rates of Re-engineering of rnr1-R503I into pol2-L439V msh2Δ
pol2 MSH2 segregants were assayed. strain recapitulates the eex phenotype.

Defects in these activities accelerate tumorigenesis due

to accumulation of mutations.

Conclusions

PPpro
Results
● Intragenic Pol2 antimutators may allow alternative
New potential antimutators within Pol ε. editing pathways to repair mismatches.

● Mutations in genes involved in the S-phase checkpoint

and dNTP synthesis lower overall mutation rate.

● Drugs that target dNTP pools may be used to modulate

Multiple defects in DNA replication drive cells to the mutator phenotype of certain cancers.
error-induced extinction (EEX).
● Future directions include further elucidating the
mechanism by which Ctf18 acts as an antimutator.
KKR is part of a highly conserved motif that
binds in the minor groove and recognizes
mismatches after they have been extended
by the polymerase. eex mutants enhance
pol2-L439V msh2Δ colonies,
the dissociation of pol E from the
mismatched DNA, allowing alternative
Acknowledgements
circled in red, show poor growth repair pathways to correct the error.
due to EEX. Grant funding provided by R01 GM118854 and R21 ES021544.