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A Role of CTCF in Recruitment of Homologous Recombination Checkpoint proteins BRCA1-BARD1 at DNA damage sites.

초록/요약

To ensure genome integrity, cells are installed with molecular machineries for DNA damage response. The toxic DNA damage, DNA double strand breaks (DSB), can be repaired by homologous recombination pathways and non-homologous end joining pathways. CCCTC-binding factor (CTCF) plays an essential role in genomic imprinting, transcription, and chromatin looping. It has been recently reported that CTCF is also involved in DNA damage responses. Here, I show that CTCF is engaged in BRCA1-BARD1 recruitment at DSB sites through its interactions with BARD1 and HP1. Depletion of CTCF impairs BRCA1-BARD1 and HP1 recruitment at damage sites. Depletion of HP1 impairs the DSB accumulation of BRCA1-BARD1 but not CTCF. In contrast, depletion of either BRCA1 or BARD1 has no effects on the recruitment of CTCF and HP1 at DSBs. Furthermore, I show that CTCF interacts with BARD1 and HP1 through its zinc finger domain. Taken together, these data indicate that CTCF acts as an essential upstream player for recruitment of HP1 and BRCA1-BARD1 at DSBs via its interactions to maintain genome integrity.

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목차

Introduction 1
Material and Methods 4
1. Cell culture 4
2. Plasmid constructs and transfection 4
3. Immuno-precipitation 5
4. Laser micro-irradiation 5
5. Immunofluorescence 5
6. Proximity Ligation Assay 6
7. Antibodies 7
Results 9
1. CTCF interacts with BARD1, but not BRCA1. 9
2. CTCF-dependent recruitment of BARD1 at DNA damage sites. 13
3. CTCF interacts with BARD1 through Zinc finger domain. 18
4. BARD1 interacts with CTCF through RING domain. 20
5. HP1 is required for BARD1-CTCF interaction upon DNA damage. 21
6. Direct interaction with HP1 is important for CTCF and BARD1 binding. 26
Discussion 28
References 31
국문요약 34

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