臺灣博碩士論文加值系統

B型肝炎病毒Hepatitis B virus屬於hepadnavridae的一員，病毒顆粒具有外套膜，而病毒基因體本身相當的精小，只有3.2 kb左右，以不完全雙股DNA存在於病毒顆粒中。當B型肝炎病毒感染肝臟細胞後，會脫去外殼將病毒基因體 (relaxed-circular DNA )釋放出來，並且在細胞核中被修補成為環狀共價鍵結DNA的型態(cccDNA)，這個cccDNA會當作模板轉錄出大小不同的mRNA ( 3.5, 2.4, 2.1, 0.6 kb ) 來合成各種不同的病毒蛋白。其中3.5 kb mRNA ( pregenomic RNA )會透過本身所轉譯出來的反轉錄酶來合成病毒的子代基因體，包裹入新的病毒顆粒，緩和的釋放到血液裡進行下一波的感染。在B型肝炎的治療上，疫苗在台灣已獲得不錯的成果，但仍有為數不少的病人必須面臨終身服用抗病毒藥物的情況，一旦停藥後，血清中的病毒量又便會上升，造成治療上的問題。而問題的根源在於細胞核中的cccDNA並沒有辦法對目前的治療產生有效反應，同時cccDNA降解速率相當的漫長，無法被清除的cccDNA就會造成病人產生持續性的感染。因此，若能夠解破cccDNA是如何從rcDNA形成的，就是提供另一種抗病毒藥物的選擇策略。而從rcDNA的結構中可以觀察到具有單股 DNA的存在，這是類似於一個DNA的損傷情況。在閱覽Base-excision repair (BER)中我們發現到了一個相當有趣的宿主蛋白, FEN1, (flap endonuclease 1)。 FEN1 是一個核酸內切酶參與在解除Okazaki fragment的RNA primer和BER的5’flap結構。我們觀察到在rcDNA的結構上也具有RNA primer和5’flap結構的存在，而在將FEN1 knockdown之後，我們發現到cccDNA的形成有20 % 顯著意義的下降。而在我們以FEN1 inhibitor, PTPD,抑制FEN1功能的情況下，我們觀察到高濃度的PTPD同時抑制了rcDNA和cccDNA的形成，同時出現一個未知的DHBV band存在；另外，在低濃度的情況下，我們觀察到cccDNA形成也有一個下降的趨勢。總結，從我們的實驗裡可以發現FEN1 確實顯著的影響cccDNA的形成，同時在inhibitor的結果可以得知，在特定的濃度下，rcDNA的形成也受到了影響而消失。

Hepatitis B virus (HBV) is an enveloped dsDNA virus belongs to hepadnaviridae. The viral genome is a partially double strand DNA form or called the relaxed circular DNA form (rcDNA), in the mature virion. After HBV entry the hepatocyte, the rcDNA will be transported into nucleus and converted to the covalently close circular DNA (cccDNA). The cccDNA persists in the nucleus and serves as template to transcript four viral mRNA, 3.5 kb pregenomicRNA (pgRNA), 2.4 kb preS RNA, 2.1 kb S RNA and 0.7 kb X RNA. The longest transcript, pgRNA, also serves as a template for viral replication and will be converted into the rcDNA by the unique reverse transcription in the nucleocpasid. In the current therapy of chronic HBV infection, the RT inhibitors can success to reduce the viral titer in the serum. However, most of the patients must take the RT inhibitor for life-long due to the persistent cccDNA in the nucleus. This cccDNA was from its precursor rcDNA with the help of some host factors. The host factors involve in the cccDNA are still unidentified now. We found a host factor, FEN1, a crucial endonuclease involved in Okazaki fragment maturation and Base-excision repair might play a role in removal of the negative-strand 5’ redundancy. In this study, we found the significant reduction of cccDNA formation by knockdown of FEN1. And when we treated with FEN1 specific inhibitor, PTPD, the cccDNA is reduced in low concentration inhibitor. However, in high concentration of inhibitor, both rcDNA and cccDNA formation were blocked. Meanwhile, an unidentified band was accumulated in high concentration of inhibitor treatement. According to our results, we found that the FEN1 significantly influent the cccDNA formation and the inhibitor data also consist with this result. And we also observe that the high concentration inhibitor block rcDNA formation. However, the detail blocking level still needs further identifications.

中文摘要 i
Abstract ii
TABLE of CONTENTS iii
LIST of FIGURES vii
1. INTRODUCTION 1
1.1 History and classification of Hepatitis B virus 1
1.2 Epidemiology and Clinical persistent 1
1.3 Virion structure 3
1.4 Genome structure and organization 4
1.5 Replication strategy and life cycle 5
1.5.1 Unique replication strategy 5
1.5.2 From pregenomic RNA to relaxed-circular DNA (Reverse transcription) 6
1.5.3 From relaxed-circular DNA to covalently-closed circular DNA 7
1.6 Duck hepatitis B virus, a good model for studying cccDNA formation of hepadnaviruses 8
1.6.1 Genome and structure of DHBV 8
1.6.2 The difference between DHBV and HBV 9
1.7 The role of DNA repair in formation of hepadnavirus cccDNA 10
1.7.1 DNA repair mechanisms 10
1.7.2 The similarity between HBV rcDNA structure and BER long patch repair pathway 13
1.8 Flap endonuclease 1 and its potential role in hepadnavirus cccDNA formation 13
1.8.1 Introduction of FEN1 13
1.8.2 Biochemical properties of FEN1 and its proposed biological roles 14
1.8.3 FEN1 structure and FEN1-like protein 17
1.8.4 The potential role of FEN1 in cccDNA formation 18
2. SPECIFIC AIM 20
3. MATERIALS AND METHODS 21
3.1 Cell lines and Cell culture system 21
3.2 Antibodies 21
3.3 Small molecule inhibitor 21
3.4 Southern blot analysis 22
3.5 DIG-labled DNA probe synthesis 23
3.6 Plasmids 24
3.7 DNA transfection 25
3.8 Modified Hirts’ extraction method 25
3.9 Alkaline preparation method 26
3.10 shRNA knockdown system 27
3.11 Total protein extraction 28
3.12 Western blotting analysis 28
4. RESULTS 30
4.1 rcDNA and cccDNA were generated from the DHBV-expression vector. 30
4.2 Knockdown of FEN1 slightly reduced the cccDNA formation of DHBV in HEK293T cells. 31
4.3 The specific FEN1 inhibitor significantly blocked the cccDNA formation. 33
4.4 Titration of the effect of PTPD on the formation of DHBV cccDNA. 34
4.5 The identity of the unknown band which was generated and accumulated after PTPD treatment. 35
5. DISSCUSIONS 38
5.1 The evidence supporting the involvement of FEN1 in regulation of DHBV cccDNA formation. 38
5.2 The inhibition step of DHBV genome replication targeted by PTPD. 40
5.3 Current studies on cccDNA formation and the other potential candidates. 41
6. FIGURES 44
7. APPENDIX 56
8. REFERENCES 57

1Blumberg, B. S. Australia Antigen and the Biology of Hepatitis B. Science (1977).
2BLUMBERG BS, A. H., VISNICH S. A "NEW" ANTIGEN IN LEUKEMIA SERA. JAMA. (1965).
3Blumberg, B. S., Gerstley, B. J., Hungerford, D. A., London, W. T. & Sutnick, A. I. A serum antigen (Australia antigen) in Down''s syndrome, leukemia, and hepatitis. Annals of internal medicine 66, 924-931 (1967).
4Dane, D. S., Cameron, C. H. & Briggs, M. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1, 695-698 (1970).
5Schaefer, S. Hepatitis B virus taxonomy and hepatitis B virus genotypes. World Journal of Gastroenterology (2006).
6Mason, A. R. J. W. Hepadnaviruses of Birds. (2008).
7WHO Media centre. Hepatitis B virus. (2008).
8WM., L. Hepatitis B virus infection. N Engl J Med. (1997).
9Jules L. Dienstag, M. D. Hepatitis B Virus Infection. The new england journal of medicine (2008).
10Heermann KH, G. U., Schwartz W, Seyffarth T, Baumgarten H, Gerlich WH. Large surface proteins of hepatitis B virus containing the pre-s sequence. J Virol. (1984).
11Kaplan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H. & Robinson, W. S. DNA polymerase associated with human hepatitis B antigen. Journal of virology 12, 995-1005 (1973).
12JESSE SUMMERS, A. O. C., AND IRVING MILLMAN. Genome of hepatitis B virus: Restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proceedings of the National Academy of Sciences of the United States of America (1975).
13Lutwick LI, R. W. DNA synthesized in the hepatitis B Dane particle DNA polymerase reaction. J Virol. (1977).
14Lien, J. M., Petcu, D. J., Aldrich, C. E. & Mason, W. S. Initiation and termination of duck hepatitis B virus DNA synthesis during virus maturation. Journal of virology 61, 3832-3840 (1987).
15Cattaneo R, W. H., Schaller H. Hepatitis B virus transcription in the infected liver. EMBO J. (1984).
16Weber M, B. V., Bartos H, Bosserhoff A, Bartenschlager R, Schaller H. Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription. J Virol. (1994).
17Wang GH, S. C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell. (1992).
18Zoulim, F., Saputelli, J. & Seeger, C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. Journal of virology 68, 2026-2030 (1994).
19Chen, H. S. et al. The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. Journal of virology 67, 1218-1226 (1993).
20Mason, J. S. a. W. S. Replication of the Genome of a Hepatitis B-Like Virus by Reverse Transcription of an RNA intermediate. Cell 29 (1982).
21Wang GH, S. C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol. (1993).
22Hirsch RC, L. J., Chang LJ, Varmus HE, Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription. Nature. (1990).
23Bartenschlager R, J.-N. M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. (1990).
24Kann M, S. A., Rabe B. Intracellular transport of hepatitis B virus. World J Gastroenterol. (2007).
25Nassal, M. Hepatitis B viruses: reverse transcription a different way. Virus research 134, 235-249, doi:10.1016/j.virusres.2007.12.024 (2008).
26Junker-Niepmann M, B. R., Schaller H. A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J. (1990).
27Zoulim F, S. C. Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. J Virol. (1994).
28Nassal M, R. A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. J Virol. (1996).
29Loeb DD, H. R., Ganem D. Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements. EMBO J. (1991).
30Haines, K. M. & Loeb, D. D. The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus. Journal of molecular biology 370, 471-480, doi:10.1016/j.jmb.2007.04.057 (2007).
31Habig, J. W. & Loeb, D. D. Sequence identity of the direct repeats, DR1 and DR2, contributes to the discrimination between primer translocation and in situ priming during replication of the duck hepatitis B virus. Journal of molecular biology 364, 32-43, doi:10.1016/j.jmb.2006.08.095 (2006).
32Staprans, S., Loeb, D. D. & Ganem, D. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. Journal of virology 65, 1255-1262 (1991).
33Yang W, M. W., Summers J. Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver. J Virol. (1996).
34Yang W, S. J. Infection of ducklings with virus particles containing linear double-stranded duck hepatitis B virus DNA: illegitimate replication and reversion. J Virol. (1998).
35Werle–Lapostolle, B. et al. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy1. Gastroenterology 126, 1750-1758, doi:10.1053/j.gastro.2004.03.018 (2004).
36Tuttleman, J. S., Pourcel, C. & Summers, J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47, 451-460 (1986).
37Dallmeier K, S. U., Nassal M. Heterologous replacement of the supposed host determining region of avihepadnaviruses: high in vivo infectivity despite low infectivity for hepatocytes. PLoS Pathog., doi:10.1371/journal.ppat.1000230.
g001 (2008).
38Schultz U, G. E., Nassal M. Duck hepatitis B virus: an invaluable model system for HBV infection. Adv Virus Res. (2004).
39Zhang, Y. Y. et al. Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proceedings of the National Academy of Sciences of the United States of America 100, 12372-12377, doi:10.1073/pnas.2033898100 (2003).
40Summers J, S. P., Horwich AL. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol. (1990).
41Lindahl, T. & Barnes, D. E. Repair of Endogenous DNA Damage. Cold Spring Harbor Symposia on Quantitative Biology 65, 127-134, doi:10.1101/sqb.2000.65.127 (2000).
42JH., H. DNA damage, aging, and cancer. N Engl J Med. (2009).
43Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Molecular cell 40, 179-204, doi:10.1016/j.molcel.2010.09.019 (2010).
44Kawanishi, S., Hiraku, Y., Pinlaor, S. & Ma, N. Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis. Biological chemistry 387, 365-372, doi:10.1515/BC.2006.049 (2006).
45Lord, C. J. & Ashworth, A. The DNA damage response and cancer therapy. Nature 481, 287-294, doi:10. 1038/nature10760 (2012).
46A., S. DNA excision repair. Annu Rev Biochem. (1996).
47T., L. Instability and decay of the primary structure of DNA. Nature. (1993).
48EC., F. DNA damage and repair. Nature. (2003).
49Neeley WL, E. J. Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem Res Toxicol. (2006).
50David SS, W. S. Chemistry of Glycosylases and Endonucleases Involved in Base-Excision Repair. Chemical reviews. (1998).
51David, S. S., O''Shea, V. L. & Kundu, S. Base-excision repair of oxidative DNA damage. Nature 447, 941-950, doi:Doi 10.1038/Nature05978 (2007).
52Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nature reviews. Molecular cell biology 11, 196-207, doi:10.1038/nrm2851 (2010).
53Lieber, M. R. NHEJ and its backup pathways in chromosomal translocations. Nature structural & molecular biology 17, 393-395, doi:10.1038/nsmb0410-393 (2010).
54Jiricny, J. The multifaceted mismatch-repair system. Nature reviews. Molecular cell biology 7, 335-346, doi:10.1038/nrm1907 (2006).
55Tomlinson, C. G., Atack, J. M., Chapados, B., Tainer, J. A. & Grasby, J. A. Substrate recognition and catalysis by flap endonucleases and related enzymes. Biochemical Society transactions 38, 433-437, doi:10.1042/BST0380433 (2010).
56Lieber, M. R. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. BioEssays : news and reviews in molecular, cellular and developmental biology 19, 233-240, doi:10.1002/bies.950190309 (1997).
57Liu, Y., Kao, H. I. & Bambara, R. A. Flap endonuclease 1: a central component of DNA metabolism. Annual review of biochemistry 73, 589-615, doi:10.1146/annurev.biochem.73.012803.092453 (2004).
58Shen, B. et al. Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases. BioEssays : news and reviews in molecular, cellular and developmental biology 27, 717-729, doi:10.1002/bies.20255 (2005).
59Bambara RA, M. R., Henricksen LA. Enzymes and Reactions at the Eukaryotic DNA Replication Fork. The Journal of biological chemistry (1997).
60Liu, Y. et al. Coordination between polymerase beta and FEN1 can modulate CAG repeat expansion. The Journal of biological chemistry 284, 28352-28366, doi:10.1074/jbc.M109.050286 (2009).
61Parrish JZ, Y. C., Shen B, Xue D. CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptotic DNA degradation. EMBO J. (2003).
62Saharia, A. et al. Flap endonuclease 1 contributes to telomere stability. Current biology : CB 18, 496-500, doi:10.1016/j.cub.2008.02.071 (2008).
63Zheng, L. et al. Fen1 mutations result in autoimmunity, chronic inflammation and cancers. Nature medicine 13, 812-819, doi:10.1038/nm1599 (2007).
64Zheng, L. et al. Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks. EMBO reports 6, 83-89, doi:10.1038/sj.embor.7400313 (2005).
65Guo, Z. et al. Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair. Molecular and cellular biology 28, 4310-4319, doi:10.1128/MCB.00200-08 (2008).
66Liu, P. et al. Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria. Molecular and cellular biology 28, 4975-4987, doi:Doi 10.1128/Mcb.00457-08 (2008).
67Hasan S, S. M., Hassa PO, Imhof R, Gehrig P, Hunziker P, Hubscher U, Hottiger MO. Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol Cell. (2001).
68Henneke, G., Koundrioukoff, S. & Hubscher, U. Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation. Oncogene 22, 4301-4313, doi:10.1038/sj.onc.1206606 (2003).
69Guo, Z. et al. Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding. Nature chemical biology 6, 766-773, doi:10.1038/nchembio.422 (2010).
70Chapados BR, H. D., Han S, Qiu J, Yelent B, Shen B, Tainer JA. Structural basis for FEN-1 substrate specificity and PCNA- mediated activation in DNA replication and repair. Cell. ( 2004).
71Qiu, J. et al. Interaction interface of human flap endonuclease-1 with its DNA substrates. The Journal of biological chemistry 279, 24394-24402, doi:10.1074/jbc.M401464200 (2004).
72Devos, J. M., Tomanicek, S. J., Jones, C. E., Nossal, N. G. & Mueser, T. C. Crystal Structure of Bacteriophage T4 5'' Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates. Journal of Biological Chemistry 282, 31713-31724, doi:10.1074/jbc.M703209200 (2007).
73Murante RS, R. L., Bambara RA. Calf 5'' to 3'' exo/endonuclease must slide from a 5'' end of the substrate to perform structure-specific cleavage. The Journal of biological chemistry (1995).
74Balakrishnan, L., Gloor, J. W. & Bambara, R. A. Reconstitution of eukaryotic lagging strand DNA replication. Methods 51, 347-357, doi:10.1016/j.ymeth.2010.02.017 (2010).
75Finger, L. D. et al. The 3''-flap pocket of human flap endonuclease 1 is critical for substrate binding and catalysis. The Journal of biological chemistry 284, 22184-22194, doi:10.1074/jbc.M109.015065 (2009).
76Harrington, J. J. & Lieber, M. R. DNA structural elements required for FEN-1 binding. The Journal of biological chemistry 270, 4503-4508 (1995).
77Murante RS, H. L., Turchi JJ, Bambara RA. The calf 5''- to 3''-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage. J Biol Chem. (1994).
78Harrington JJ, L. M. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. (1994).
79Liu, R., Qiu, J., Finger, L. D., Zheng, L. & Shen, B. The DNA-protein interaction modes of FEN-1 with gap substrates and their implication in preventing duplication mutations. Nucleic acids research 34, 1772-1784, doi:10.1093/nar/gkl106 (2006).
80Zheng, L. et al. Functional regulation of FEN1 nuclease and its link to cancer. Nucleic acids research 39, 781-794, doi:10.1093/nar/gkq884 (2011).
81Kucherlapati, M. et al. Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression. Proceedings of the National Academy of Sciences of the United States of America 99, 9924-9929, doi:10.1073/pnas.152321699 (2002).
82Larsen, E., Gran, C., Saether, B. E., Seeberg, E. & Klungland, A. Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst Stage. Molecular and cellular biology 23, 5346-5353, doi:10.1128/mcb.23.15.5346-5353.2003 (2003).
83Zheng, L., Dai, H., Qiu, J., Huang, Q. & Shen, B. Disruption of the FEN-1/PCNA interaction results in DNA replication defects, pulmonary hypoplasia, pancytopenia, and newborn lethality in mice. Molecular and cellular biology 27, 3176-3186, doi:10.1128/MCB.01652-06 (2007).
84Klungland A, L. T. Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. (1997).
85Liu, Y. et al. DNA polymerase beta and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair. The Journal of biological chemistry 280, 3665-3674, doi:10.1074/jbc.M412922200 (2005).
86Parenteau J, W. R. Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease. Genetics. (2002).
87Sampathi, S., Bhusari, A., Shen, B. & Chai, W. Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance. The Journal of biological chemistry 284, 3682-3690, doi:10.1074/jbc.M805362200 (2009).
88Saharia, A. & Stewart, S. A. FEN1 contributes to telomere stability in ALT-positive tumor cells. Oncogene 28, 1162-1167, doi:10.1038/onc.2008.458 (2009).
89Storici F, H. G., Ferrari E, Gordenin DA, Hubscher U, Resnick MA. The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair. The EMBO journal (2002).
90Friedrich-Heineken, E., Henneke, G., Ferrari, E. & Hubscher, U. The Acetylatable Lysines of Human Fen1 are Important for Endo- and Exonuclease Activities. Journal of molecular biology 328, 73-84, doi:10.1016/s0022-2836(03)00270-5 (2003).
91Kao, H. I., Henricksen, L. A., Liu, Y. & Bambara, R. A. Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. The Journal of biological chemistry 277, 14379-14389, doi:10.1074/jbc.M110662200 (2002).
92Harrington, J. J. & Lieber, M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes & development 8, 1344-1355, doi:10.1101/gad.8.11.1344 (1994).
93Lieber, M. R. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. (1997).
94Shen B, Q. J., Hosfield D, Tainer JA. Flap endonuclease homologs in archaebacteria exist as independent proteins. (1998).
95Kanai, Y. et al. DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork. The FEBS journal 274, 3914-3927, doi:10.1111/j.1742-4658.2007.05924.x (2007).
96Ip, S. C. et al. Identification of Holliday junction resolvases from humans and yeast. Nature 456, 357-361, doi:10.1038/nature07470 (2008).
97West, S. C. The search for a human Holliday junction resolvase. Biochemical Society transactions 37, 519-526, doi:10.1042/BST0370519 (2009).
98Hohl, M., Thorel, F., Clarkson, S. G. & Scharer, O. D. Structural determinants for substrate binding and catalysis by the structure-specific endonuclease XPG. The Journal of biological chemistry 278, 19500-19508, doi:10.1074/jbc.M213155200 (2003).
99Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071-1078, doi:10.1038/nature08467 (2009).
100Zhu, Y., Wu, Z., Cardoso, M. C. & Parris, D. S. Processing of lagging-strand intermediates in vitro by herpes simplex virus type 1 DNA polymerase. Journal of virology 84, 7459-7472, doi:10.1128/JVI.01875-09 (2010).
101Tumey, L. N. et al. The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors. Bioorganic & medicinal chemistry letters 15, 277-281, doi:10.1016/j.bmcl.2004.10.086 (2005).
102Dorjsuren, D., Kim, D., Maloney, D. J., Wilson, D. M., 3rd & Simeonov, A. Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity. Nucleic acids research 39, e11, doi:10.1093/nar/gkq1082 (2011).
103Condreay LD, A. C., Coates L, Mason WS, Wu TT. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J Virol. (1990).
104Guo, J. T. et al. Conditional replication of duck hepatitis B virus in hepatoma cells. Journal of virology 77, 1885-1893 (2003).
105Wu, T. T., Coates, L., Aldrich, C. E., Summers, J. & Mason, W. S. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175, 255-261 (1990).
106Asagoshi, K. et al. FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells. Molecular cancer research : MCR 8, 204-215, doi:10.1158/1541-7786.MCR-09-0253 (2010).
107Matsuzaki Y, A. N., Koyama H. Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2. Nucleic Acids Res. (2002).
108Moe, S. E., Sorbo, J. G. & Holen, T. Huntingtin triplet-repeat locus is stable under long-term Fen1 knockdown in human cells. Journal of neuroscience methods 171, 233-238, doi:10.1016/j.jneumeth.2008.03.012 (2008).
109Hohl, M. et al. Domain swapping between FEN-1 and XPG defines regions in XPG that mediate nucleotide excision repair activity and substrate specificity. Nucleic acids research 35, 3053-3063, doi:10.1093/nar/gkm092 (2007).