ISG15에 의한 PCNA의 변형이 Error-Prone Translesion DNA Synthesis의 종결에 미치는 영향에 관한 연구

Abstract

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2014. 8. 정진하.In response to DNA damage, PCNA is mono-ubiquitinated and triggers translesion DNA synthesis (TLS) by recruiting polymerase-. However, it remained unknown how error-prone TLS is turned off after DNA lesion bypass to prevent mutagenesis. Here, I showed that ISG15 modification (ISGylation) of PCNA plays a key role in TLS termination. Upon UV irradiation, EFP, an ISG15 E3 ligase, bound to mono-ubiquitinated PCNA and promoted its ISGylation. ISGylated PCNA then tethered USP10 for deubiquitination and in turn the release of polymerase- from PCNA. Eventually, PCNA was deISGylated by UBP43 for reloading of replicative DNA polymerases and resuming normal DNA replication. However, ISGylation-defective Lys-to-Arg mutations in PCNA or knockdown of any of ISG15, EFP, or USP10 led to persistent recruitment of mono-ubiquitinated PCNA and polymerase- to nuclear foci, causing an increase in mutation frequency and in turn a decrease in cell survival. These findings establish a crucial role of PCNA ISGylation in termination of error-prone TLS for preventing excessive mutagenesis and thereby for the maintenance of genome stability.TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS iii LIST OF FIGURES AND TABLES vi BACKGROUND 1 1. Translesion DNA synthesis (TLS) 1 2. Proliferating cell nuclear antigen (PCNA) 2 3. Ubiquitin 4 4. Interferon-stimulated gene 15 (ISG15) 5 5. Purpose of thesis work 11 INTRODUCTION 12 MATERIALS AND METHODS 17 1. Plasmids and antibodies 17 2. Cell culture and transfection 18 3. Immunoprecipitation and NTA pull-down analysis 19 4. Purification of proteins 19 5. Immunocytochemistry 20 6. Chromatin fractionation 20 7. supF plasmid-based mutation assay 21 8. Cell cycle analysis 22 RESULTS 23 1. UV induces ISG15-conjugating system 23 2. PCNA has two ISG15 acceptor sites 28 3. UV induces sequential modification of PCNA by ubiquitin and ISG15 39 4. EFP serves as an ISG15 E3 ligase of PCNA 52 5. Mono-ubiquitination of PCNA is required for its interaction with EFP 59 6. USP10 has a PIP box and removes mono-ubiquitin from PCNA 64 7. ISGylation of PCNA is required for its interaction with USP10 74 8. PCNA ISGylation causes the release of Pol form PCNA 77 9. PCNA ISGylation down-regulates nuclear foci formation 80 10. PCNA ISGylation blocks TLS-mediated mutagenesis 85 11. PCNA is eventually deISGylated by UBP43 104 DISCUSSION 115 REFERENCES 122 ABSTRACT IN KOREAN 133Docto