呂勝春臺灣大學:分子醫學研究所張雅玲Chang, Ya-LingYa-LingChang2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51351PARP-1是一個參與在DNA修復、轉錄、複製、重組以及調節染色質結構的多弁鉬簿嚏C FACT是一個由FACTp140和SSRP1組成的核蛋白。FACT已知在轉錄染色質模板時扮演著延長因子角色。在過去的研究中顯示PARP-1和FACT存在同一個多重蛋白複合體中。而在我們的研究中,我們發現PARP-1和FACT間的交互作用。它們之間的交互作用主要是透過PARP-1與FACTp140作用而不是透過SSRP1。PARP-1 與FACT間的交互作用是有細胞週期性的,且其作用最好的時期是在S phase。此外,它們的交互作用也可以被 g-射線刺激增加。FACTp140會增強PARP-1的酵素活性。綜合而言,我們證明了FACT可以調節PARP-1的酵素活性,而這樣的調節對於PARP-1在DNA複製、轉錄以及DNA修復有一定的重要性。Poly(ADP-ribose) polymerase-1 (PARP-1) is a multifunctional enzyme involving in DNA repair, transcription, replication, recombination and modulation of chromatin structure. FACT is a nuclear protein composed of FACTp140 and SSRP1 heterodimeric complex. FACT is a known chromatin-specific elongation factor required for transcription of chromatin templates. Previous studies have shown that PARP-1 and FACT exist in the same multiprotein complex. In this study the interaction between PARP-1 and the FACT was examined. PARP-1 interacts with FACT through FACTp140, not SSRP1. The interaction between PARP-1 and FACT is cell-cycle-dependent, peaks at S phase. The interaction is stimulated after g-irradiation. FACTp140 may stimulate the automodification activity of PARP-1. In conclusion, we demonstrate that FACT can regulate the enzyme activity of PARP-1, an event important for PARP-1’s function in replication, transcription and DNA repair.Abstract (Chinese) 1 Abstract (English) 2 Introduction 3 Materials and Methods Cell culture 7 Preparation of whole cell extract 7 Cell synchronization 8 Irradiation procedure 8 Antibodies 9 Immunoprecipitation and western blot analysis 9 In vitro pull down assay 10 PARP poly(ADP-ribosyl)ation assay using purified proteins 11 Preparation of cell nuclear extract 11 Gel-filtration fractionation 12 Plasmids and constructs 13 Expression of recombinant PARP-1 and FACTp140 13 Results FACT associated with PARP-1 through its subunit FACTp140 15 Determination of PARP-FACT multiprotein complex 15 Cell-cycle dependent interaction between PARP-1 and FACT 16 The alteration of PARP-FACT complex after g–irradiation 16 FACT may stimulate the enzyme activity of PARP-1 17 Discussion 19 Figures Figure 1 22 Figure 2 23 Figure 3 24 Figure 4 25 Figure 5 26 Figure 6 27 Figure 7 28 Figure 8 29 Figure 9 30 Figure 10 31 Figure 11 32 Figure 12 33 References 34 Appendix 421414736 bytesapplication/pdfen-US交互作用PARP研究探討PARP-1與FACT的交互作用Functional studies of interaction between PARP-1 and FACTotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51351/1/ntu-93-R91448001-1.pdf