RPA (Replication protein A) 是一種核內單股DNA結合蛋白，廣泛地表現在真核細胞，其主要是由RPA1、RPA2及RPA3三個次體組合而成一穩定複合物存在。目前研究所知，RPA的功能主要和DNA複製、修補以及基因重組有關，但除了和單股DNA結合作用外，RPA亦會與其他蛋白有交互作用的發生。例如，過去研究顯示RPA1可與SV40T antigen、DNA polymerase a、VP16及p53結合；而RPA2亦可與XPA、RAD52及uracil-DNA glycosylase等參與DNA修補相關之蛋白結合。此外，RPA2在細胞週期G1/S phase時期有磷酸化現象，直到M phase晚期時則進行去磷酸化作用。當細胞受到外來刺激 (如UV激活)，致使細胞核內DNA損害進行修補時，RPA會受到來自細胞內的一些kinase作用而有高度磷酸化的現象。研究指出複製作用進行時的DNA損傷會導致DNA-PK (DNA-dependent protein kinase) 磷酸化RPA2，而此磷酸化作用可能傳遞了DNA損害之訊息並連結至S-phase check point機制，使p53從RPA-p53複合物中釋出，進一步而導致p53之活化。由上述結果可看出RPA2及其磷酸化現象在DNA進行代謝過程或在調控細胞週期上的重要性。然而就目前所知，RPA2的生理角色仍未清楚了解。為了釐清及探討RPA2在細胞中及其在生理上可能角色之扮演，我們嘗試找出其他會與RPA2結合作用的蛋白，並希望從而更能了解RPA2磷酸化與其他結合蛋白之交互作用的生理意義。本實驗室先前利用Yeast-Two Hybrid之方法，成功地由人類乳腺細胞基因庫中，篩選出一個會與RPA2直接交互作用的蛋白4E-BP3。4E-BP3為一個分子量14.6 kDa之蛋白，屬於4E-BPs家族的一員。過去研究發現去磷酸化的4E-BPs會與eIF4G競爭eIF4E的結合位置，使得cap-mRNA無法帶入43S pre-initiation complex，因而抑制轉譯的速率，這樣的作用對於細胞生長有負向調節的功能。然而，我們證實RPA2與4E-BP3兩者蛋白質有直接交互作用的結果，顯示RPA2與4E-BP3可能在細胞中參與調控彼此的功能。在本實驗中，為了更加了解RPA2與4E-BP3之間交互作用的情形，利用免疫沉澱法證實在HEK293T及HepG2細胞中，RPA2與4E-BP3會形成複合物。進一步利用免疫細胞染色法、共軛焦顯微鏡觀察以及核內蛋白質萃取，結果發現RPA2會特異性的表現在細胞核內，而4E-BP3則是均勻的分布在整個細胞中，兩者間的交互作用會發生在細胞核內。另外，實驗結果亦觀察到RPA2與4E-BP3之間是屬於專一性的的結合，而RPA1並不參與兩者間的交互作用。利用細胞受曝射UV的刺激模式，可以觀察到RPA2會有磷酸化的現象，而磷酸化態的RPA2不會與4E-BP3相互結合，而且去磷酸化酵素抑制劑的有無亦不影響兩者之間的交互作用，推測RPA2與4E-BP3的結合並非受蛋白質磷酸化與否所調控。同時，根據細胞曝射UV後不同時間點的觀察顯示兩種蛋白失去結合現象發生的相當快，在細胞經UV刺激後十分鐘即產生變化，這樣的結果可能是來自於UV照射激活細胞而產生DNA損害的訊息，所以使得HEK293T及HepG2細胞內RPA2與4E-BP3之間原本的結合作用消失。而在細胞同步化的實驗結果發現RPA2與4E-BP3的相互結合會受到細胞週期所調控，在S phase時，RPA2與4E-BP3的結合表現較G0/G1、G2/M phase弱。因此，我們認為會和4E-BP3交互作用的RPA2應該是原本就會基本表現於細胞內的游離態RPA2，而當細胞進行DNA複製或DNA修補時，RPA2會和RPA1、RPA3一起組合成RPA複合物參與細胞生理功能的維持，所以我們可以觀察到在UV照射之後或是在S phase單股DNA存在時，RPA2與4E-BP3的結合相對就減少了。綜合言之，本論文的實驗結果證明了細胞核內RPA2與4E-BP3之間會相互結合，而此兩種蛋白的交互結合與分開的現象不但會受到細胞週期所調控，同時還可能參與在細胞受UV激活損害的生理調控機轉上。

RPA (replication protein A), a ubiquitous eukaryotic single-stranded DNA binding protein complex, that is composed of RPA1, RPA2 and RPA3 subunits. The heterotrimeric complex has a high affinity for single stranded and damage DNA, and plays essential role in DNA replication, repair and recombination. Furthermore, RPA function is mediated by its phosphorylation and protein-protein interactions. RPA2 is phosphorylated in a cell cycle dependent manner and is additionally phosphorylated in response to DNA damage, such as UV or ionizing irradiation, and treatment with replication inhibitors. The phosphorylation of RPA2 may elicit the signaling transduction during DNA damage and link to the S-phase checkpoint mechanism. The purpose of our study is to identify the RPA2 binding proteins and elucidate the biochemical and functional characteristics of RPA2 phosphorylation. In our previous studies, a RPA2 binding protein, 4E-BP3 (eukaryotic translation initiation factor 4E-binding protein 3), has been selected from the genome library of human gland mammalian cell line by the Yeast-Two hybrid technique. The 4E-BP3, a member of 4E-BPs family, is a 14.6 kDa protein that is able to compete with eIF4G for binding to eIF4E (eukaryotic initiation factor 4E), thereby stifling eIF4F complex-directed translation. In this study, RPA2 interacts with 4E-BP3 but not with 4E-BP1 and 4E-BP3 interacts with RPA2 but not with RPA1 were demonstrated by using the Yeast-Two-Hybrid method and immunoprecipitation assay. The results of immunohistochemistry staining and confocal microscopy assay revealed RPA2 localized in the nucleus of HEK293T, while 4E-BP3 localized in both cytoplasm and nucleus. Further experiments using immunoprecipitation showed that RPA2-4E-BP3 complex was found in the nuclear fraction. These data indicated that the interaction of RPA2 with 4E-BP3 may occur only in the nucleus. In UV-stimulated cells, the phosphorylated form of RPA2 was detected in 2 hours later. When HEK293T cell or HepG2 cell was irradiated with 50 J/m2 of UV, dissociation of RPA2 and 4E-BP3 was detected at 10 minute and no RPA2 and 4E-BP3 complexes was detected at 30 minute. We purpose the loss of binding ability of 4E-BP3 for RPA2 in HEK293T and HepG2 cell lines may due to the signaling of DNA damage induced by UV stimulation. Cells serum starvation or aphidicolin blocking, the interaction of RPA2 with 4E-BP3 was in a cell cycle dependent manner. The binding level in S phase was weaker than G0/G1 or G2/M phase. We purpose 4E-BP3 may interact with free RPA2 but not with RPA complex. When RPA2 forms RPA complex with RPA1 and RPA3 in S phase or after UV damage, it will dissociated with 4E-BP3.Taken together, the results showed RPA2 would interacts with 4E-BP3 in nuclear. The interaction and de-association of RPA2 and 4E-BP3 may involve in the physiological regulation of cell cycle and cell responses to UV damage.

Abromova, et al., 1997. Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner. Proc. Natl. Acad. Sci. USA 94:7186-7191.

Adachi, et al, 1992. Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. J. Cell Biol. 119: 1-15.

Arnim, et al., 1994. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5’-cap function. Nature 371, 762~767.

Blackwell, et al., 1996. Single-stranded DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase. Mol. Cell. Biol. 16: 4798-4806.

Bochkarev, et al., 1999. The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J. 18: 4498-4504.

Braun, et al., 1999. Manuscript in preparation.

Brunn G. J., et al., 1996. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 15 (19): 5256-5267.

Brush, et al., 1994. The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication. Proc. Natl. Acad. Sci. USA 91: 12520-12524.

Bushell M., et al., 1999. Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis. FEBS Lett. 451: 332-336.

Cardoso, et al., 1993. Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell 74: 979-792.

Carty, et al., 1992. HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro. Mutat. Res. DNA Repair 274: 29-43.

De Benedetti, et al., 1999. eIF4E expression in tumours. Int J Biochem Cell Biol 31:59-72.

De Benedetti, et al., 1994 CHO cells transformed by the translation factor eIF4E display increased c-myc expression, but require overexpression of Max for tumorigenicity .Mol Cell Diff 2:347-391.

Denis, et al., 1996. The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth. Oncogene 13: 2415-2420.

Din, et al., 1990. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 4: 968-977.

Dutta, et al., 1992. Cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 11: 2189-2199.

Elevated, et al., 1995. Levels of Eukaryotic translation initiation factor eIF4E mRNA in a borad sepectrum of transformed cell line. Cancer Lett 91:247-252.

Fleurent, et al., 1997. Angiotensin II stimulates phosphorylation of the translational repressor 4E-binding protein 1 by a mitogen-activated protein kinase-independent mechanism. J. Biol. Chem. 272(7): 4006-4012.

Francis, et al., 1998. 4E-BP3, a new member of the Eukaryotic initation factor 4E-binding protein family. J. Biol. Chem. 273:14002-14007.

Georgaki, et al., 1993. DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit. Nucleic. Acids Res. 21: 3659-3665.

Gingras, et al., 1999. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes & Development 13 (11):1422-1437.

Gingras AC. Raught B. Sonenberg N., 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annual Review of Biochemistry. 68:913-963.

Graves L. M, et al., 1995. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc. Natl. Acad. Sci USA 92 (16):7222-7226.

Gomes, et al., 1995. Structural analysis of human replication protein A. Mapping functional domains of the 70 kDa subunit. J. Biol. Chem. 270: 4534-4543.

Gomes, et al., 1996. Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Biochemistry 35: 5586-5595.

Gregory G., et al., 2001. UV-induced hyperphosphorylation of replication protein A depends on DNA replication and expression of ATM protein. Mol. Cell. Biol. 12:1199-1213.

Haghighat A, et al., 1995. Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO Journal. 14 (22):5701-5709.

He, et al., 1995. RPA involvement in the damage recognition and incision steps of nucleotide excision repair. Nature 374: 566-569.

Henricksen, et al., 1994. Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication. J. Biol. Chem. 269: 1121-1132.

Henricksen, et al., 1996. Phosphorylation of human replication protein A by the DNA dependent protein kinase DNA dependent protein kinase is involved in the modulation of DNA replication. Nucl. Acids Res. 24: 433-440.

Jang S. K., et al., 1998. Heterogeneous nuclear ribonucleoprotein L interacts with the 3' border of the internal ribosomal entry site of hepatitis C virus. Journal of Viology. 72 (11):8782-8788.

Kerekatte, et al., 1995. The proto-oncogene/translation factor eIF4E: a survey of its expression in breast carcinomas. Int J Cancer 1995,64:27-31.

Kenny, et al., 1989. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proc. Natl. Acad. Sci. USA 86: 9757-9761.

Kim, et al., 1996. Protective effect of ginseng on radiation-induced DNA double strand breaks and repair in murine lymphocytes. Cancer Biother Radiopharm. 11: 267-272.

Kozak M, et al., 1991.An alalysis of vertebrate mRNA sequence:initiations of translation control .Journal of Cell Biology. 115 (4):887-903.

Kate J. et al., 2001. Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1). Current Biology 11:1374-1397.

Lazaris, et al., 1990. Malignant transformation by a eukaryotic translation initiation factor subunit that binds to mRNA cap. Nature 345:544-547.

Lee S-H, Kim, D. K., 1995. Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication. J Biol Chem. 270: 12801-12807.

Lin. et al., 1995. Control of PHAS-I by insulin in 3T3-L1 adipocytes synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway. J. Biol. Chem. 270 (31):18531-18538.

Lin, et al., 1996. Dissection of functional domains of the human DNA replication protein complex replication protein A. J. Biol. Chem. 271: 17190-17198.

Liu, et al., 1993. The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cell. Mol. Cell. Biol. 13: 7222-7231.

Mader S., et al., 1995 The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Molecular & Cellular Biology. 15 (9): 4990-4997.

Marc S., et al., 1997. Replication Protein A: A Heterotrimeric, Single-Stranded DNA-Binding protein Required for Eukaryotic DNA Metabolism. Annu. Rev. Biochem. 66:61-92.

Matsunaga, et al., 1996. Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease. J. Biol. Chem. 271: 11047-11050.

Meyer, R.R. and Laine, P. S., 1990. The single-stranded DNA-binding protein of E. coli. Microbiological Reviews. 54(4): 342-380.

Marissen W., et al., 2000. Identification of caspase 3-mediated cleavage and functional alternation of eukaryotic initiation factor 2α in apoptosis. J. Biol. Chem. 275:9314-9323.

Nahum, et al., 1998. The mRNA 5’-cap-binding protein eIF4E and control of cell growth. Current opinion in Cell Biology 10:268-275.

Niu, et al., 1997. Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro. J. Biol. Chem. 272: 12634-12641.

Pan, et al., 1994. Phosphorylation of p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent-protein kinase. Proc. Natl. Acad. Sci. USA 91: 8343-8347.

Park, et al., 1996. Physical interaction between haman RAD52 and RPA is require for homologous recombination in mammalian cells. J. Biol. Chem. 271: 22111-22116.

Pain V M, et al., 1996. Initiation of protein synthesis in eukaryotic cells. European Journal of Biochemistry. 236 (3):747-771.

Pelletier and Sonenberg, 1988. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 334 (6180):320-325.

Pfuetzner, et al. 1997. Replication protein A. Characterization and crystallization of the DNA binding domain. J. Biol. Chem. 272: 430-434.

Philipova, et al., 1996. A hierarchy of SSB protomers in replication protein A. Genes Dev. 10: 2222-2233.

Pieler. T. and Rudt. F., 1997. Semin. Cell. Dev. Biol. 8: 79-82.

Salmond. G P C. and P. J. Reeves., 1993. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem. Sci. 18:7-12.

Seo, et al., 1991. Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity. J. Biol Chem. 266: 13161-13170.

Shao, et al., 1999. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA: DNA-PK complexes. EMBO J. 18: 1397-1406.

Tee, A. R., et al., 2000 DNA damage causes inactivation of translation regulators linked to mTOR signaling. Oncogene. 19:3021-3031.

Tee, A. R., et al., 2002 Caspase cleavage of initiation factor 4e-binding protein 1 yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory motif. Mol. Cell. Biol. 22.6. :1674-1683.

Von Manteuffel S R, et al., 1996. 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Proc. Natl. Acad. Sci USA 93(9): 4076-4080.
.
Wobbe, et al., 1987. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc. Natl. Acad. Sci. USA 84: 1834-1838.

Wold, M. S. et al., 1997. Replication protein A: A heterotrimeric, single-stranded DNA-binding protein require for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66: 61-92.

申屠子萍, 2001. 篩選與RPA2交互作用之蛋白質及其研究。國立成功大學醫學院生物化學研究所碩士論文。