Title Page
ABSTRACT
Contents
LIST OF ABBREVIATIONS 15
1. Introduction 17
1.1. Research background 17
1.1.1. DNA double-strand break response in mammalian cells 17
1.1.2. DNA end resection in homologous recombination repair 18
1.1.3. Modification of the MRN complex in DSB response 19
1.1.4. RECQL4 and other RECQ family helicases in DSB response 19
1.2. Purpose of Research 22
2. Materials and Methods 24
2.1. Cell culture and reagents 24
2.2. Plasmids preparation 25
2.3. Immunofluorescence staining 26
2.4. Laser micro-irradiation and real-time imaging of fluorescent proteins 27
2.5. Immunoprecipitation and immunoblotting 27
2.6. Ubiquitination assay 28
2.7. Protein stability assay 29
2.8. Homologous recombination repair assay 29
2.9. WST-1 assay for cell viability 29
2.10. Statistics analysis 30
2.11. Antibodies 30
3. Results 32
3.1. Stable maintenance of the MRN complex is sufficient to restore the DNA DSB response in cells lacking RECQL4 helicase activity. 32
3.1.1. The MRN complex is prematurely disassembled from DSB sites in RTS and RECQL4-depleted cells. 32
3.1.2. The helicase activity of RECQL4 is required for stable maintenance of the MRN complex on DSB sites during DSB response. 37
3.1.3. SCFSKP2-dependent ubiquitination is responsible for premature disassembly of the MRN complex from DSB sites.[이미지참조] 41
3.1.4. K48-linked ubiquitination of NBS1 by SCFSKP2 E3 ligase increases in cells with DSBs.[이미지참조] 45
3.1.5. Stable maintenance of the MRN complex is sufficient to restore the DSB response in RECQL4-defective cells. 50
3.2. USP2 stabilizes the MRN complex at DNA DSB sites by counteracting the ubiquitination of NBS. 56
3.2.1. Overexpression of USP2 restores the DSB response in RECQL4-defective cells. 56
3.2.2. USP2 is recruited to DSB sites and counteracts the ubiquitination of NBS1. 61
3.2.3. USP2 is recruited to DSB sites in a manner dependent upon ATM, PARP, and RECQL4. 66
3.2.4. The N-terminus of USP2 is sufficient for ATM-dependent recruitment to DSB sites. 73
3.2.5. Phosphorylation of two critical residues in USP2 N-terminus is required for its recruitment to DSB sites and interaction with RECQL4. 78
3.2.6. USP2 and USP28 play a redundant role in the stable maintenance of the MRN complex at DSB sites during the DSB response. 84
3.2.7. p97/VCP activity is required for dissociation of the MRN complex from DSB sites. 93
4. Discussion 95
4.1. K48-linked ubiquitination of NBS1 is critical for disassembly of the MRN complex. 97
4.2. USP28 overexpression recovered repair defects in RECQL4-defective cells, but depletion of USP28 hardly affected DSB response. 98
4.3. USP2 plays a redundant role with USP28 in counteracting NBS1 ubiquitination in the DSB response. 100
4.4. Counteracting continued NBS1 ubiquitination by deubiquitinases may prevent unwanted accumulation of the MRN complex. 103
4.5. USP2 may indirectly reduce NBS1 ubiquitination through interaction with SKP2. 104
4.6. USP2 has a potential to influence the MRN stability in the absence of RECQL4. 105
4.7. RECQL4 and its helicase activity may indirectly play a role in DSB response. 106
4.8. Mouse and human cells showed differences in defects caused by loss of RECQL4. 109
4.9. Differences in the expression level of deubiqutinases in different tissues can be the reason why most of the symptoms of RTS are concentrated in certain tissues. 110
5. References 111
Abstract in Korean 119
Table 1. Antibodies used in this study. 31
Table 2. Relative expression levels of USP2 and USP28 in U2OS, HELA, or H460 cell lines found in Cancer Cell Line Encyclopedia. 102
Figure 1. The MRN complex is prematurely disassembled from DSB sites in RECQL4-depleted U2OS cells. 33
Figure 2. EGFP-fused MRE11 is prematurely dissociated from DSB sites in RECQL4-depleted cells. 35
Figure 3. Instability of the MRN complex in RTS cells is recovered by expression of RECQL4. 36
Figure 4. Stable maintenance of the MRN complex on DSB sites requires helicase activity of RECQL4. 40
Figure 5. Premature disassembly of the MRN complex is recovered by inhibition of proteasome. 42
Figure 6. SCFSKP2-dependent ubiquitination is responsible for premature disassembly of the MRN complex.[이미지참조] 44
Figure 7. The s tability of NBS1 protein is decreased in RECQL4-depleted cells treated with NCS. 46
Figure 8. SCFSKP2-dependent ubiquitination of NBS1 occurs in cells treated with NCS.[이미지참조] 47
Figure 9. SCFSKP2-dependent ubiquitination of NBS1 is prevented by lysine-to-arginine substitution at amino acid residue 735.[이미지참조] 49
Figure 10. Defects in DSB response are restored by the expression of USP28 in RECQL4-defective cells. 51
Figure 11. The s tability of the MRN complex on double-strand break sites is restored in RTS AG18371 cells by overexpression of USP28. 52
Figure 12. Expression of USP28 as well as RECQL4 decreases sensitivity of RTS cells to DSB inducing reagents. 53
Figure 13. USP28 counteracts the ubiquitination of NBS1 to stabilize the MRN complex. 55
Figure 14. Overexpression of USP2 restores the DSB response in RECQL4-depleted U2OS cells. 57
Figure 15. Overexpression of USP2 restores the HR ability in RECQL4-depleted cells. 59
Figure 16. Overexpression of USP2 restores the DSB response in RTS cells. 60
Figure 17. USP2 is recruited to laser-induced DSB sites. 62
Figure 18. USP2 interacts with NBS1. 63
Figure 19. USP2 counteracts the ubiquitination of NBS1. 65
Figure 20. USP2 is recruited to DSB sites in a manner dependent on ATM. 67
Figure 21. USP2 is recruited to DSB sites in a manner dependent on PARylation. 70
Figure 22. USP2 is recruited to DSB sites in a manner dependent on RECQL4. 71
Figure 23. USP2 interacts with RECQL4 in a manner dependent on DSB and ATM. 72
Figure 24. USP2 binds to DSB sites through its N-terminus. 74
Figure 25. N-terminus of USP2 is recruited to DSB sites in a manner dependent on PARylation and ATM. 75
Figure 26. N-terminus of USP2 is recruited to DSB sites in a manner dependent on RECQL4. 76
Figure 27. N-terminus of USP2 interacts with RECQL4 in a manner dependent on DSB. 77
Figure 28. Phosphorylation of two critical residues in USP2 N-terminus by ATM is required for recruitment to DSB sites. 80
Figure 29. Phospho-mimetic mutant USP2 is recruited to DSB sites, but this recruitment is prevented by inhibition of ATM. 81
Figure 30. Phosphorylation of two critical residues in USP2 N-terminus by ATM is required for interaction with RECQL4. 83
Figure 31. Inactivation of both USP2 and USP28 activities results in decrease of EGFP-MRE11 recruitment to micro-irradiation site. 86
Figure 32. Inactivation of both USP2 and USP28 activities results in premature disassembly of the MRN complex. 87
Figure 33. NBS1 ubiquitination is prevented by inactivation of both USP2 and USP28 activities. 89
Figure 34. NBS1 protein stability is decreased by inactivation of both USP2 and USP28 activities. 90
Figure 35. Inactivation of both USP2 and USP28 activities results in defects in ATM activation and HR repair abilities. 92
Figure 36. Inhibition of p97/VCP prevents premature disassembly of the MRN complex from DSB sites. 94
Figure 37. Diagram showing the control of NBS1 ubiquitination during the DSB response. 96