Survival of DNA Damage in Yeast Directly Depends on Increased dNTP Levels Allowed by Relaxed Feedbac

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Survival of DNA Damage in Yeast Directly Depends
on Increased dNTP Levels Allowed by Relaxed
Feedback Inhibition of Ribonucleotide Reductase

• Mary Heaton
• November 15, 2004

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DNA Damage Response
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Ribonucleotide Reductase

• Enzyme that catalyzes the production of dNTPs for
  DNA replication and DNA damage repair
• Mammalian RNR is characterized in detail but
  there is homology to other eukaryotic,
  prokaryotic, and viral systems
• Regulated several ways including
• Allosteric regulation
• Transcriptional regulation
• Inhibition by Sml1

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Structure of RNR

• RNR is an a2ß2 heterotetramer consisting of an R1
  and an R2 subunit
• R1-allosteric sites to regulate dNTP pool balance
  and overall dNTP levels
• R2-binuclear ferric iron centers with tyrosyl
  free radicals (involved in catalysis)

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RNR Genes

• RNR1 codes for large subunit essential
• RNR2 codes for small subunit essential
• RNR3 found in yeast to code for large subunit
  not essential but studies have shown that
  overexpression of RNR3 compensates for an RNR1
  null allele
• RNR4 codes for an R2 like protein believed to
  help fold rnr2p and stabilize it for formation of
  free radical centers

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Regulation of RNR in Saccharomyces cerevisiae

• Transcriptional Regulation
• CRT1(RFX1) recruits the general repressors Ssn6
  and Tup1 to bind to the promoter of RNR genes
  (removed by DUN1)
• Inhibition by Sml1
• Binds to R1 subunit but is removed by mec1/rad53
  during DNA damage (also decreased levels during S
  phase)
• Feedback Inhibition
• dNTPs bind to R1 subunit to balance dNTP pools
  and regulate overall activity of enzyme by
  ATP/dATP ratio

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Regulation of RNR in Saccharomyces cerevisiae
dNTPs
DNA break
NTPs
active RNR enzyme
Microfilament protein complex
Sml1
inactive RNR enzyme
RNR genes
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In the cell cycle

• Increase in activity during S phase because Sml1
  concentrations are lower, possibly to allow for
  DNA replication
• Also increase in activity during DNA damage
  because Sml1 is degraded

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Problems

• Few studies have investigated the affect of
  regulation on dNTP pools in vivo
• All three mechanisms work together to regulate
  RNR, but it is not known which one is the primary
  regulator
• It is unclear if dNTP levels increase after
  damage RNR activity likely increases but dNTP
  levels are supposed to repress it

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Questions

• Why is yeast RNR not allosterically regulated by
  dATP?
• Previous study showed RNR to not be regulated by
  dATP in vitro at levels up to 50 µM (mammals
  5-10µM) relaxed feedback inhibition
• What will happen in vivo?
• Is RNR activity regulated primarily Sml1 and
  transcriptional regulation?
• Will dNTP levels increase?

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Increase in dNTP levels?

• First, wild-type and rnr3? yeast treated with
  4-nitroquinoline-N-oxide (acts like UV), methyl
  methane sulfonate (DNA alkylating agent), and UV
  light
• 6-8 fold increase after 2.5 hours

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Increase in dNTP levels?
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What happens in an RNR mutant?

• Constructed an RNR mutant that no longer
  contained an allosteric site for dATP inhibition,
  rnr1-D57N
• Idea derived from studies on mouse R1 protein, a
  homolog of Rnr1 and Rnr3
• Change aspartic acid to asparagine in position 57
  and dATP allosteric site is destroyed
• Tests confirm that mutant is completely resistant
  to dATP

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Testing normal growth

• No obvious phenotype
• dNTP pools are 1.6-2-fold higher compared to wt
  (although not as dramatic increase as in
  mammalian cells)

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What are dNTP levels when damage is induced?

• Treated rnr1-D57N mutant and wt with
  4-NQO
• Mutant cells showed 20-30 fold increase in dNTP
  levels over untreated wt and a 4-fold increase
  over 4-NQO treated wt
• Therefore, dATP feedback is active during DNA
  damage!

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What are dNTP levels when damage is induced?
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Biological Effect

• Because of increased dNTP pools, rnr1-D57N
  mutants show more resistance to UV light, 4-NQO,
  and MMS compared to wt
• However, there is also a 2-fold increase in
  mutation rates compared to wt
• Specific increase in G?C and G?T transversions
  and frameshift insertions

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Contribution of Each Regulatory Mechanism

• Mutation made in each regulatory pathway
• sml1?
• dun1? for transcriptional regulation
• rnr1-D57N
• dun1? exhibits severe DNA damage sensitivity
• Sensitivity equal to wt levels in dun1? rnr1-D57N
  and dun1 ? sml1?
• sml1? shows no significant increase in survival
  over wt
• Increased survival in sml1? rnr1-D57N equal to
  rnr1-D57N
• Interestingly, dun1 ? sml1? rnr1-D57N exhibits
  high resistance to damage like rnr1-D57N

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Biological Effect
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dNTP Levels in S Phase vs. DNA Damage

• Increase in purine dNTPs by 6-fold and pyrimidine
  dNTPs by 3-fold during S phase
• NTP pools do not fluctuate during cell cycle
• dNTP pools in logarithmically growing wt cells
  induced by 4-NQO are 3-5 fold higher than in
  untreated S phase cells

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Conclusions
DNA break
NTPs
active RNR enzyme
Microfilament protein complex
Sml1
inactive RNR enzyme
RNR genes
RNR genes
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Conclusions

• RNR machinery designed to provide different dNTP
  levels during cell cycle and after DNA damage
• Relaxed dATP feedback inhibition allows increase
  in levels after damage
• Once dNTP levels are high enough, dATP feedback
  inhibition kicks in

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Conclusions

• Direct correlation between increased dNTP levels
  and survival during damage
• Increased dNTP levels also correlate to increased
  mutation rates
• Polymerases are more likely to make mistakes at
  dG and dC bases in presence of higher dNTP
  concentrations (no pool imbalance)

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Does this happen in mammalian cells?

• No clear answers yet
• Strict dATP inhibition of mammalian RNR
• However, increased RNR activity does not result
  in an increase in dNTP levels
• But this was done in absence of DNA damage
• Mammalian cells may have relaxed dATP feedback
  mechanism that is unknown
• Further studies in mammals should be conducted

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References

• Chabes, A., Georgieva, B., Domkin, V., Zhao, X.,
  Rothstein, R., Thelander, L. (2003). Survival of
  DNA damage in yeast directly depends on increased
  dNTP levels allowed by relaxed feedback
  inhibition of ribonucleotide reductase. Cell.
  112, 391-401.
• Elledge, S.J., Zhou, Z., Allen, J.B. (1992).
  Ribonucleotide reductase regulation, regulation,
  regulation. Trends Biochem. Sci. 17, 119-123.
• Huang, M. Zhou, Z., Elledge, S.J. (1998). The DNA
  replication and damage checkpoint pathways induce
  transcription by inhibition of the Crt1
  repressor. Cell. 94, 595-605.
• Zhou, B.S., Elledge, S. (2000). The DNA damage
  response putting checkpoints in perspective.
  Nature. 408, 433-439.
• Zhao, X., Chabes, A., Domkin, V., Thelander, L.,
  Rothstein, R. The ribonucleotide reductase
  inhibitor Sml1 is a new target of the Mec1/Rad53
  kinase cascade during growth and in response to
  DNA damage. EMBO J. 20, 3544-3553.