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

****

**** *

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