1. New players in the BRCA1-dependent
DNA damage responses
성균관대학교 생명과학과
김 홍 태

2. Checkpoint activation
DNA repair

3. Cell cycle checkpoints are a self-defense
system
Internal hazards:
Oxidative species,
DNA replication errors,
Mitotic errors…
External hazards:
UV, Ionizing radiation,
Chemicals ...
Damage
Cell cycle checkpoint
Loss of checkpoints
Stop cell cycle
Continue cell cycle
Fix the lesions
Genomic instability
Apoptosis
Genomic stability
Cancer

4. Outline of DNA damage induced Cell Cycle Checkpoints
DNA strands breaks
Sensors
ATM, ATR
BRCA1
53BP1
MDC1
Topbp1
Mediators/
Transducers
Effectors/Kinases
Chk1, Chk2
Apoptosis
DNA repair
Checkpoint
control

5. BRCA1 (breast cancer susceptibility gene 1)
1863
Ring domain
NLS
BRCT domain
( Ub E3 ligase)
a. ~ 50% familial breast cancer patients and ~30% familial
ovarian cancer patients carry BRCA1 mutations.
--Functions as a tumor suppressor
b. Conditional Knockout mice – Increase the tumor formation
2. BRCA1 participates in DNA damage response.
Translocate to DNA damage sites

6. BRCA1 forms nuclear foci following
DNA damage
gH2AX
DNA damage
BRCA1
(Ionizing Radiation) ?
BRCA1
DAPI
(pH2AX) _ IR
IR: ionizing radiation (5Gy)

7. BRCA1 foci localization depends on
its BRCT domain
HCC1937
HCC1937
BRCA1
BRCT
HCC1937
BRCA1
HCC1937
DNA damage
(IR, 5Gy)

8. Which proteins recruit the BRCA1
at the DNA damage site?
Ring domain
BRCT domain pS
NLS
BACH1, CtIP
SPXF
SPTF—BACH1
SPVF-- CtIP

9. RAP80 is another BRCA1 interaction protein
(high affinity to streptavidin, can be eluted by biotin)
S tag
SBP
BRCA1 BRCT
2xFlag
Lysates from K562 cells
stably expressing BRCT
BACH1
CtIP
RAP80
Streptavidin
beads
S beads
Eluted with
biotin
Eluted with
detergent
* Indicates non-specific bands

10. Introduction of RAP80 (Retinoid receptor
Associated Protein 80) 1
719
UIMs
Zinc finger domain
RAP80 is a nuclear protein
Interacts with retinoid-related testis-associated
receptor in vitro
Transcription repression
No reports on the RAP80 is involved in the DNA
damage checkpoint or binds to the BRCA1

11. Binding assay data between BRCA1 and RAP80

12. Does RAP80 relocalize to the DNA break sites
and colocalize with BRCA1 after exposure to
ionizing radiation?
Using immunofluorescense assay
Biochemical assay

13. RAP80 and H2AX IF data in 293T cells

14. RAP80 and BRCA1 IF data in 293T cells

15. Which regions are important for the RAP80 DNA foci formation?
Using immunofluorescense assay

16. RAP80 WT and serial deletion mutants IF data

17. Alignments of the UIM containing proteins

18. RAP80 WT and point mutants IF data

19. Only RAP80 UIMs are involved in the DNA foci formation
HSJ1a UIM1
HSJ1a UIM2
UIM conserved PXXAX XX SXXAc core

20. GST-pull down assay data

21. Summary I 1. RAP80 UIM is important its relocalization on the DNA
damage sites.
2. RAP80 UIM directly binds to the Lys-63 and
Lys-6 poly-Ub chains

22. Hierarchy in the IR-induced DNA damage response pathway
IR-induced DNA strands breaks
ATM
rH2AX
The position of RAP80 ?
MDC1
BRCA1
NBS1
53BP1

23. Does RAP80 DNA foci formation depend on the
MDC1, -H2AX, NBS1, 53BP1 and BRCA1
after exposure to IR?
Using immunofluorescense assay

24. RAP80 and H2AX IF data in the NBS1and 53BP1 -/- and +/+ cells

25. RAP80 and H2AX IF data in the BRCA1and H2AX -/- and +/+ cells

26. in the MDC1 -/- and +/+ cells
RAP80 and H2AX IF data
in the MDC1 -/- and +/+ cells

27. BRCA1, MDC1 and H2AX IF data in the RAP80 -/- and +/+ cells

29. Hierarchy in the IR-induced DNA damage response pathway
ATM/ATR
pH2AX
MDC1
RAP80
BRCA1
53BP1

30. Is RAP80 involved in the BRCA1-dependent checkpoint
control?
IR-induced cell survival assay
IR-induced G2/M checkpoint analysis
IR-induced CHK1 phosphorylation

31. IR-induced cell survival assay

32. IR-induced G2/M checkpoint analysis

35. RAP80 x
Phosphorylation
dependent
pS/pT
BRCA1
BRCT

36. Introduction of CCDC98 (Coiled-Coil Domain
-Containing protein 98) 1
409
Coiled-Coil Domain
Novel protein
Only contains coiled-coil Domain

37. Binding assay data

38. RAP80 x
Phosphorylation
dependent p
BRCA1
BRCT

39. CCDC98 binds to BRCA1-BRCT on phosphorylation
dependent manner

40. The sequences of the CCDC98 A.A
MEGESTSAVLSGFVLGALAFQHLNTDSDTEGFLLGEVKGEAKNS
ITDSQMDDVEVVYTIDIQKYIPCYQL FSFYNSSGEVNEQALKKILS
NVKKNVVGWYKFRRHSDQIMTFRERLLHKNLQEHFSNQDLVFLL
LTPSII TESCSTHRLEHSLYKPQKGLFHRVPLVVANLGMSEQLGY
KTVSGSCMSTGFSRAVQTHSSKFFEEDGSLK EVHKINEMYASLQ
EELKSICKKVEDSEQAVDKLVKDVNRLKREIEKRRGAQIQAAREK
NIQKDPQENIFL CQALRTFFPNSEFLHSCVMSLKNRHVSKSSCN
YNHHLDVVDNLTLMVEHTDIPEASPASTPQIIKHKALD LDDRWQ
FKRSRLLDTQDKRSKADTGSSNQDKASKMSSPETDEEIEKMKG
FGEYSRSPTF
SPTF BACH1
SPVF CTIP

41. CCDC98 SPTF sequence is important for binding
With BRCA1-BRCT

42. Check the CCDC98 phosphorylation

43. CCDC98, BRCA1 and H2AX IF data in 293T cells

44. RAP80 x
Phosphorylation
dependent p
BRCA1
BRCT

45. Requirement of CCDC98 for damage-induced BRCA1 foci formation

46. CCDC98 WT and serial deletion mutants IF data
in the 293T cells

47. Binding assay data between CCDC98
and BRCA1 or RAP80

48. RAP80 (235-337 A.A) is important for binding
With CCDC98

49. IR-induced Chk1 phosphorylation and cell survival assay

50. IR-induced G2/M checkpoint analysis

51. Conclusion
1. RAP80 was identified as a new BRCA1-BRCT binding protein.
2. CCDC98 was identified as a mediator between BRCA1 and RAP80.
3. UIM regions of RAP80 directly bound to ubiquitin and is important for
the relocalization of RAP80 to the DNA damage sites.
4. RAP80 functions in the BRCA1 upstream
5. CCDC98 and RAP80 are involved in the IR-induced G2/M checkpoint and –cell
survival assay.

52. Checkpoint activation
DNA repair