Maintenance of Genomic Integrity by RSF1 Chromatin Remodeler

PART1 1 I. INTRODUCTION 2 II. MATERIALS AND METHODS 4 A. Cell culture and treatments 4 B. Generation of RSF1 KO cells 4 C. Transfection and RNA interference 4 D. Plasmid and purification of recombinant proteins 5 E. Immunoprecipitation and in vitro binding assay 5 F. In vitro kinase assay 6 G. Cell fractionation 6 H. Chromosome spreading 7 I. Immunocytochemistry 7 J. Live cell imaging 7 III. RESULTS 9 A. RSF1 localizes at mitotic kinetochores and directly interacts with PLK1 9 B. RSF1 plays a crucial role in PLK1 deposition and activation at the mitotic kinetochores 16 C. Phosphorylation of RSF1 at Ser1375 by CDK1 is necessary and sufficient for PLK1 docking to kinetochores 22 D. PLK1-dependent secondary phosphorylation of RSF1 contributes to stable deposition of PLK1 at the kinetochores 27 E. RSF1-deficient cells suffering from chromosome-alignment defects are rescued by phosphomimetic RSF1 mutants, but not by non-phosphorylatable mutants 31 IV. DISCUSSION 35 PART2. 43 I. INTRODUCTION 44 II. MATERIALS AND METHODS 46 K. Immunoprecipitation and in vitro binding assay 46 L. Cell fractionation 46 M. Chromosome spreading 46 III. RESULTS 48 F. RSF1 gradually disappears from chromosome arms and accumulates at centromeres upon chromosome condensation 48 G. RSF1 directly interacts with HDAC1 but, not HP1α during mitosis 50 H. RSF1 plays a crucial role in HDAC1 deposition at the mitotic chromosome arms 53 I. Knockdown of RSF1 causes defects in sister-chromatid cohesion and kinetochore protein assembly 56 J. C-terminal of RSF1 is necessary and sufficient for protection of centromeric cohesion 58 K. HDAC1 dissociation from chromosome arm is essential for the translocation of HP1α in RSF1 depleted cells 60 IV. DISCUSSION 63 V. REFERENCES 66 VI. 국문요약 73

Author dc:creator

• 이, 호수

Contributors dc:contributor

• 조, 혜성
• 대학원 의생명과학과
• 107400

• Chromatin remodeler
• RSF1
• mitosis
• PLK1
• HDAC1

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000021909
000000021909

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/13041