Title Page
ABSTRACT
Contents
LIST OF ABBREVIATIONS 8
1. Introduction 10
2. Materials and Methods 18
2.1. Cell culture and cell lines 18
2.2. Plasmid and siRNA transfection 18
2.3. Plasmids 18
2.4. siRNAs 18
2.5. iPOND 18
2.6. Immunoprecipitation and western blot analysis 19
2.7. Antibodies 19
2.8. AP-MS analysis 20
2.9. CUPID analysis 20
2.10. Protein purification 20
2.11. An in vitro pull-down assay 21
2.12. Chromatin fractionation 21
2.13. An EU incorporation assay 22
2.14. QUANTIFICATION AND STATISTICAL ANALYSIS 22
3. Results 23
3.1. ATAD5 is enriched on nascent DNA and BET proteins interact with ATAD5 23
3.2. The ET domain of BET proteins interacts with ATAD5 26
3.3. ATAD5 (596-692) is an ET domain-binding motif. 29
3.4. ATAD5 directly interacts with BRD4 through its ET domain-binding motif 32
3.5. BRD4 binding to ATAD5 does not interfere with mRNA transcription 35
3.6. Inhibition of BRD4-acetylated histone binding decreases the amount of chromatin bound PCNA 38
3.7. The level of chromatin bound PCNA is regulated by ATAD5-BRD4 interaction 40
4. Discussion 44
REFERENCES 46
Figure 1. Eukaryotic replication fork structure and the roles of PCNA 13
Figure 2. PCNA loading and unloading is mediated by RFC and RFC-like-complexes 15
Figure 3. PCNA unloading by ATAD5-RLC is crucial for maintaining genomic integrity 17
Figure 4. ATAD5 is enriched on nascent DNA and BET proteins interact with the N-terminal... 25
Figure 5. The ET domain of BET proteins is crucial for the interaction with ATAD5 28
Figure 6. Conserved β-stranded structure of ATAD5 is a BRD4 ET domain-binding motif 31
Figure 7. ATAD5 directly interacts with BRD4 through its ET domain-binding motif 34
Figure 8. BRD4 binding to ATAD5 does not interfere with global mRNA transcription 37
Figure 9. Inhibition of BRD4-acetylated histone binding decreases the amount of nascent... 39
Figure 10. The level of chromatin bound PCNA is regulated by ATAD5-BRD4 interaction 42
Figure 11. BRD4 fine-tunes the PCNA unloading activity of ATAD5-RLC 43