Tatiana MoiseevaPostdoctoral associate
Chris Bakkenist lab @ University of Pittsburgh
DNA repair interest group videoconferenceApril 24th 2018
ATR/Chk1 axis controls replication origin firing
DNA replication initiation
active origins
dormant origins
Pol α
Pol ε
Cdc45
GINSAnd-1
Pol δ
MCM helicase
replicationfork
Cdk2Cyclin
E/A
Cdc7 Dbf4
kinases responsible for replication
initiation
ATM/ATR-dependent replication checkpoint
Cdc25A
Cdk2Cyclin
E/ACdc7 Dbf4
?
Chk2T68 Chk1 S345
S317
ATM ATR
double strand break
single stranded DNAreplication stress
replication initiation
DNA damage normal replication ATR inhibition
ATR
activ
ityATR activity and replication
Kwok et al., 2015
Model
ATR
Chk1
origin firing
ATRi Normal replication
Cdc7
How does Chk1 activity regulate Cdc7 activity?
Which origins are these?
ssDNA
MCM4
cont
rol
ATRi
Moiseeva et al., Nature Communications, 2017
Replication proteins recruited to chromatin
And-1/WDHD1
GINS
Which origins fire in response to ATR inhibition?
• Are ATRi effects specific to early or late origins?
• Is ATRi affecting the replication timing program (late origins would fire in early S-phase)?
ATR inhibition → increased EdU incorporation throughout the S-phase
early late
ATRi equally affects early and late S-phase cells
U2OS
DMSO/ATRi EdU
30’ 45’
ATR inhibition → origins firing in the vicinity of ongoing replication
ldU, 15’ +/-ATRi, 15’ CldU, 5’
ATR inhibition does not affect replication timing program
How does ATR/Chk1 activity prevent Cdc7-dependent origin firing?
Chk1
Cdc7 at dormant origins
Dormant origin firing
?
Model: Rif1-PP1 interaction is stabilized by phosphorylation
Rif1PP1
pChk1pRif1
Cdc7
Chk1
Cdc7at dormant
origins
ssDNA, fork slowing
Chk1
Cdc7at dormant
origins
Hiraga et al., 2017:
Disruption of Rif1/PP1 interaction causes increased origin firing, fork stalling, MCM4 phosphorylation on chromatin.
Rif1(PP1-interacting motif)
Rif1-PP1 interaction is blocked by ATR/Chk1 inhibition
Rif1-PP1 interaction is blocked by ATR/Chk1 inhibition
****
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Proximity Ligation Assay
Rif1-PP1 interaction is inhibited by λ−phosphatase treatment
ATR
Chk1
ATRi Normal replication
Cdc7 at dormant origins
Rif1PP1
p
1. ATR/Chk1 activity blocks dormant origin firing throughout S-phase.
2. ATR inhibition does not affect replication timing program.
3. Rif1 interaction with PP1 phosphatase is blocked by ATRi or Chk1i.
4. Rif1 interaction with PP1 phosphatase is phosphorylation dependent.
5. Rif1 interaction with PP1 phosphatase is blocked by S2205 mutations.
ATR/Chk1 activity stops dormant origins from firing by localizing PP1 phosphatase to
Rif1 protein to reverse Cdc7-dependent phosphorylations
Conclusions:
local dormant origin firing
University of Pittsburgh
Chris Bakkenist, PhDSandy Schamus for all the mutations and constructs all Bakkenist lab members and neighbors
Genome stability group
Inova Schar Cancer Institute, Annandale, VA
Thomas Conrads, PhDBrian Hood, PhD
mass-spectrometry
THANK YOU!
Acknowledgements
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