Title Page
Contents
ABSTRACT 12
I. Identification of a novel factor implicated in DNA double strand break repair and causative genetic alterations leading to the hereditary genetic disorder, Fanconi anemia 16
1. Introduction 17
2. Materials and Methods 22
3. RESULTS 28
3.1. Mutation analysis of pwwp2b mutations of stomach adenocarcinoma 28
3.2. The PWWP2B is identified as a novel UHRF1 binding protein 31
3.3. PWWP2B plays important roles in DNA double strand break repair pathway 33
3.4. End resection is severely impaired in cells depleted with PWWP2B 36
3.5. PWWP2B is associated with MRE11 38
3.6. PWWP2B is require for ATM activation upon DNA damage 41
3.7. PWWP2B is required for gastric cancer cell proliferation 43
3.8. Identification of novel fanca mutation in Korean Fanconi anemia patients 45
3.9. Molecular and cellular analysis confirmed pathogenicity of the mutation 48
3.10. Complementation analysis of patient cells with wild-type FANCA 51
II. Development of therapeutic strategy for Fanconi anemia through CRISPR-Cas9 mediated gene editing 54
1. Introduction 55
2. Materials and Methods 57
3. Results 59
3.1. Establishment of fanca gene editing strategy in Korean Fanconi anemia patient cell 59
3.2. The efficiency of CRISPR-Cas9 in Korean Fanconi anemia patients' cell lines 61
3.3. Restoration of FANCA expression in single cell from Korean Fanconi anemia patient' knock-out cells 65
3.4. MMC stimulation increases edited population in knock-out cells 74
III. Discussion 78
IV. REFERENCES 82
ABSTRACT IN KOREAN 88
Table 1. siRNA sequences used in Chapter I. 27
Table 2. characteristics of individuals with Fanconi anemia and... 46
Figure 1. Mutation analysis of PWWP2B mutations of stomach adenocarcinoma 29
Figure 2. PWWP2B is identified as a novel UHRF1 binding protein 32
Figure 3. PWWP2B is implicated in DDR and HR-mediated DSB repair 34
Figure 4. PWWP2B participates in DNA end resection during HR 37
Figure 5. PWWP2B is associated with MRE11 40
Figure 6. PWWP2B is required for ATM activation upon DNA damage 42
Figure 7. PWWP2B depletion sensitize cells to PARP inhibitor in 44
Figure 8. Allele specific sequence analysis identifies novel FANCA... 47
Figure 9. Molecular and cellular characterization of cell lines from... 49
Figure 10. Complementation of KFA5 and KFA6 cells with the... 53
Figure 11. schematic diagram of FANCA gene editing strategy 60
Figure 12. The efficiency of CRISPR-Cas9 in Korean Fanconi anemia... 63
Figure 13. The efficiency of gene editing using CRPSPR-Cas9 in... 64
Figure 14. FANCA was detected after KFA5-7KO single cell isolation 67
Figure 15. FANCA was detected after KFA5-7KI single cell isolation 69
Figure 16. FANCA was detected after KFA5-8KO single cell isolation 71
Figure 17. FANCA was detected after KFA5-8KI single cell isolation 72
Figure 18. The schematic diagram of MMC stimulation in CRISPR-... 75
Figure 19. Edited cell proportion was increased after MMC... 76
Figure 20. Edited cell proportion was increased after MMC... 77