Cyclin D1 plays a direct role in DNA repair by interacting with proteins involved in homologous recombination. It was found to directly interact with and help recruit RAD51 to DNA damage sites, facilitating efficient DNA repair. Cyclin D1 also interacts with the C-terminus of BRCA2, helping recruit RAD51 by preventing inhibitory phosphorylation of BRCA2 by cyclin A/CDK2. Depletion of cyclin D1 impairs RAD51 focus formation and homologous recombination after DNA damage. These findings establish a novel function of cyclin D1 in promoting accurate DNA repair through homologous recombination.

1. Siwanon Jirawatnotai, Ph.D.
Siriraj Laboratory of Systems Pharmacology,
Dept of Pharmacology, Siriraj Medical School
A Direct Role of Cyclin D1 in DNA Repair

2. Cyclin D1 is a cell cycle protein that drives mammalian cell division
gallus.reactome.org

3. A function of cyclin D1 in DNA repair uncovered by proteomic screenings
TAP Mass-spec
MCF-7,
ZR-75-1
UMSCC-2
GRANTA
519
HT29
Jirawatnotai, Nature 2011

4. Cyclin D1 is required for cell survival after DNA damage
0 h 16 h
sicont
siD1-B 0
20
40
60
80
Percentofcells
Tail length (pixels)
sicont
siD1-B
0
20
40
60
80
Percentofcells
Tail length (pixels)
***
*
***
0 h 16 h

5. Pierce A .,et al. 1999
0
20
40
60
80
100
120
Percent
***
0
20
40
60
80
100
120
Percent
***
Interaction between cyclin D1 and RAD51 facilitates efficient homologous recombination
Valerie et al., 2003
Oncogene

6. Cyclin D1 directly interacts with RAD51
SP49
Z138C
Granta 519
H2009
ZR-75-1
HT-29
IP
RAD IgG WCL
WB: cyclin D1
MCF7
In vivo bindingIn vitro binding

7. N-terminus domain of cyclin D1 and C-terminus domain of RAD51 are require for the
interaction
***

8. Interaction between cyclin D1 and RAD51 facilitates RAD51 loading to damage foci
0
20
40
60
80
100
0-2 3-9 10-19
Percentofcells
Foci/nucleus
4 h
***
***
***
***
*
*
0
20
40
60
80
100
0-2 3-9 10-19
Percentofcells
Foci/nucleus
0 h
0
20
40
60
80
100
0-2 3-9 10-19
Percentofcells
Foci/nucleus
2 h
*
***
***
***
***
*
0
20
40
60
80
100
0-2 3-9 10-19
Percentofcells
Foci/nucleus
8 h sicont
siD1-A
siD1-C
***
***
***
***
**
**
0
20
40
60
80
100
0-2 3-9 10-19
Percentofcells
Foci/nucleus
6 h
***
***
***
***
***
***
sicont
siD1-A
siD1-C
DAPI RAD51 -H2AX Merge
sicont
siD1-A
siD1-C
+ DNA damage

9. Cyclin D1 localizes at damaged DNA
0
2
4
6
8
Foldenrichment
Before DNA break
After DNA break
0
2
4
6
8
Foldenrichment
siD1
0
5
10
15
20
25
IgG Cyclin
D1
E2F4
Foldenrichment
Before DNA break
After DNA break
0
5
10
15
20
25
Foldenrichment
siD1
Pierce A .,et al. 1999
Targeted ChIP
DAPI - H2AX cyclin D1 Merge
Immunofluorescence

10. Interaction between cyclin D1 and BRCA2
B2-1
B2-3
B2-4
B2-5
B2-6
B2-7
B2-8
B2-9
B2-2
GST
Cyclin D1 binding to BRCA2 in vitro
(GST-pull-down)
BRCA2+/-H2AX+
siD1-A
sicont
Esashi, et al. 2005
Input GST
RAD51 binding to BRCA2 in vitro
(GST-pull-down)
Esashi, et al. 2005
Cyclin A/CDK2 phosphorylation
of BRCA2

11. Nocodazole
D1
GST-B2-9
Roscovitin +---
-+--
++++
Cyclin A
GST-B2-9
RAD51
Cyclin D1
GST B2-9
pull-down
293 cells
Phospho
BRCA2 S3291 Kinase assay
Kinase
assay
Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by
preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2
Cyclin D1 expression helps
recruiting RAD51 to C-ter of
BRCA2
Phospho BRCA2 S3291
BRCA2
Cyclin D1
Cyclin B1
Cyclin A2
RAD51
CDK4
b-Actin
Async. G2/M (nocodazole)
- D1 Ro - D1 Ro
Cyclin B1 long exp
293 cells
Cyclin D1 expression prevents
B2-9 phosphorylation by CDK2
Lysate +++-

12. G2/M
-
Phospho BRCA2 S3291
Cyclin D1
Cyclin A2
CDK4
Cyclin B1
RAD51
HeLa cells
Expression of cyclin D1 helps recruit RAD51 to C-terminus domain of BRCA2 by
preventing the inhibitory S3291 phosphorylation by cyclin A/CDK2
Depletion of cyclin D1 by siRNA
increases S3291 phosphorylation

13. IP: B2-9
G2
IR (5 Gy) - + - +
Cyclin A2
Cyclin D1
BRCA2S3291
Asynchronous
GST B2-9
Cyclin D1 is recruited to C-terminus of BRCA2 upon DNA damage
WB

14. In vitro Competition assay
rHA-Cyclin D1
GST-BRCA2 C-Ter (B2-9) - + + + + + - + + + + +
rCyclin - - - -
+ + + + + + + + + + + +
DA
WB: HA
GST
Cyclin D1 controls phosphorylation of C-terminus BRCA2 by competing
with cyclin A for BRCA2 binding

15. Cyclin D1 cooperates with DNA damage to inhibit BRCA2
phosphorylation at Ser3291
Nocodazole - + - + - + - + - +
IR - - - - - - + + + +
D1 - - - - + + - - + +
Ros - - + + - - - - - -
1 2 3 4 5 6 7 8 9 10
S3291 Ph
cyclin D1
BRCA2
CDK4
actin
IB:
γH2AX
cyclin D1
IB:
GST
GST pulldown
0.5 1 2 4 6
Hour after IR
actin
IB:
S3291 Ph
BRCA2
cdc25a
NoIR

16. aa 20-90 of cyclin D1 is required for BRCA2-C-terminal binding
full-length
20-295
91-295
full-length
20-295
91-295
B2-5 B2-9 inp
IP
WB: Cyclin D1
full-length
WB:GST
B2-9 binding domain of cyclin D1 (aa 20-91) is required
to prevent CDK2/Cyclin A2 phosphorylation on B2-9
D1 1-295 1-90
IB:
GST
cyclin D1
B2-9 kinase assay
cyclin A-CDK2
0
20
40
60
80
100
120
0 nM 10 nM 20 nM 40 nM
Relativephosphorylationlevel
Concentration of recombinant cyclin D1
D1
del.91-
295
cyclin D1
Δ1-90
BSAB
S
A
BSA BSA

17. Cyclin box domain of cyclin D1 is required for RAD51 and BRCA2 binding
Cyclin box

18. Shot exp.
Long exp.
Shot exp.
Long exp.
Shot exp.
Long exp.
GST-B2-5
GST-B2-9
GST-pRb
GST-B2-9
GST-pRb
GST-B2-5
WB: GST
Cyclin D1 that is in complex with CDK4 does not compete with CDK2/cyclin A
Shot exp.
Long exp.
Kinase
assay
CDK4/Cyclin D1 CDK2/Cyclin A2
CDK2/Cyclin A2 CDK4/Cyclin D1
GST-B2-9
GST-Cyclin D1
GST-Cyclin A
WB: GST
1
10
100
0 Gy 2 Gy 4 Gy 6 GyPercentsurvival
0 µM
1 µM
3 µM

19. Cyclin D1 depletion hypersensitizes Rb-negative cancers to IR treatment
Jirawatnotai, Nature 2011

20. Cyclin D1 promotes RAD51 localization on damage
DNA, and HR
Jirawatnotai, Oncogene, in press

21. No DNA
damage
Severe DNA damage
Jirawatnotai, Cancer Research, 2012

24. Thanks
• Chonvara Chalermrujinanant
• Gunya Sittithumcharee
• Peter Sicinski, DFCI
– Wojtek Michowski
– Lisa Becks
• Fumiko Esashi (University of Oxford)
• David Livingston lab, DFCI
– Yiduo Hu
• Steve Gygi lab, HMS
– Joshua Elias
• John Quackenbush, CCCB, DFCI
– Yaoyu Wang
• Bing Xia (University of Medicine and Dentistry
• of New Jersey)