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

Checkpoint activation DNA repair

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

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

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

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

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

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

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

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

Binding assay data between BRCA1 and RAP80

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

RAP80 and H2AX IF data in 293T cells

RAP80 and BRCA1 IF data in 293T cells

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

RAP80 WT and serial deletion mutants IF data

Alignments of the UIM containing proteins

RAP80 WT and point mutants IF data

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

GST-pull down assay data

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

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

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

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

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

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

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

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

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

IR-induced cell survival assay

IR-induced G2/M checkpoint analysis

RAP80 x Phosphorylation dependent pS/pT BRCA1 BRCT

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

Binding assay data

RAP80 x Phosphorylation dependent p BRCA1 BRCT

CCDC98 binds to BRCA1-BRCT on phosphorylation dependent manner

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

CCDC98 SPTF sequence is important for binding With BRCA1-BRCT

Check the CCDC98 phosphorylation

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

RAP80 x Phosphorylation dependent p BRCA1 BRCT

Requirement of CCDC98 for damage-induced BRCA1 foci formation

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

Binding assay data between CCDC98 and BRCA1 or RAP80

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

IR-induced Chk1 phosphorylation and cell survival assay

IR-induced G2/M checkpoint analysis

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.

Checkpoint activation DNA repair