1 New players in the BRCA1-dependentDNA damage responses 성균관대학교 생명과학과 김 홍 태

2 Checkpoint activationDNA repair

3 Cell cycle checkpoints are a self-defensesystem 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 CheckpointsDNA 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 followingDNA damage gH2AX DNA damage BRCA1 (Ionizing Radiation) ? BRCA1 DAPI (pH2AX) _ IR IR: ionizing radiation (5Gy)

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

8 Which proteins recruit the BRCA1at 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 receptorAssociated 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 formationHSJ1a 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 DNAdamage 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 pathwayIR-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 +/+ cellsRAP80 and H2AX IF data in the MDC1 -/- and +/+ cells

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

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

30 Is RAP80 involved in the BRCA1-dependent checkpointcontrol? 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

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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 phosphorylationdependent manner

40 The sequences of the CCDC98 A.AMEGESTSAVLSGFVLGALAFQHLNTDSDTEGFLLGEVKGEAKNS 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 bindingWith 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 datain the 293T cells

47 Binding assay data between CCDC98and BRCA1 or RAP80

48 RAP80 (235-337 A.A) is important for bindingWith 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 activationDNA repair