Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth
博士 === 國立臺灣大學 === 微生物研究所 === 87 === Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase), which possesses both endonuclease and exonuclease activities and utilizes both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. Previously, studies of EBV DNase have...
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博士 === 國立臺灣大學 === 微生物研究所 === 87 === Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase), which possesses both endonuclease and exonuclease activities and utilizes both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. Previously, studies of EBV DNase have been focused on the detection of anti-DNase antibodies in the patients with EBV associated diseases, the biochemical properties and the relationship between function and structure of the enzyme. In this dissertation, one monoclonal antibody (mAb) to EBV DNase was generated and the relationship of function and structure of EBV DNase including DNA binding domains and nuclear localization signals (NLSs) was further investigated and the effect of EBV DNase on the cell growth was examined. A mAb to EBV DNase, designated 311H, was obtained and the mAb can recognize the EBV DNase protein specifically. The antigenic epitope of 311H was located to a.a. 1-152 of EBV DNase. This mAb is useful for following studies of EBV DNase. To map regions of EBV DNase responsible for DNA binding activity, a series of mutant DNase polypeptides was expressed using an in vitro transcription/translation system and their DNA binding activities to DNA cellulose were determined. The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding. However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities. In the N-terminus, deletion of residues 23-28 and residues 7-61 re sulted in the loss of nuclease activity but the DNA binding activities of the trucated enzymes were intermediate and low, respectively. Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected. DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA. These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of intact and highly ordered structure(s) for both activities. It has been reported that EBV DNase may be detected in the nucleus and/or cytoplasm of infected cells. In this study, using cell-fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (-Gal). The results demonstr ated that two regions of the DNase with NLS activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the -Gal to the nuclei of HEp-2 cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS. In NLS-B, the histidine residue at amino acid 306 is required for NLS-B activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure. EBV DNase was expresed under inducible control of lactose operon to investigate its effects on cells. Induction of DNase expression with IPTG in Raj i cells caused DNA strand breaks, which were examined by using a comet assay. In addition, the expression of DNase resulted in decreased growth of Raji cells with cell cycle arrest at G1 and accumulation of cyclin E and p27Kip1. The results imply that EBV-DNase-induced DNA strand breaks may be associated with the chromosomal instability of cells.
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author2 |
Chen Jen-Yang |
author_facet |
Chen Jen-Yang Liu Ming-Tsan 劉銘燦 |
author |
Liu Ming-Tsan 劉銘燦 |
spellingShingle |
Liu Ming-Tsan 劉銘燦 Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
author_sort |
Liu Ming-Tsan |
title |
Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
title_short |
Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
title_full |
Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
title_fullStr |
Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
title_full_unstemmed |
Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth |
title_sort |
studies on epstein-barr virus dnase: analysis of dna binding regeions and nuclear localization signals and its effects on cell growth |
publishDate |
1998 |
url |
http://ndltd.ncl.edu.tw/handle/07420301810491738708 |
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ndltd-TW-087NTU003810022016-02-01T04:12:40Z http://ndltd.ncl.edu.tw/handle/07420301810491738708 Studies on Epstein-Barr Virus DNase: Analysis of DNA Binding Regeions and Nuclear Localization Signals and Its Effects on Cell Growth EB病毒DNA分解之研究:DNA結合區域與核定位訊號的分析及其對細胞的作用 Liu Ming-Tsan 劉銘燦 博士 國立臺灣大學 微生物研究所 87 Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase), which possesses both endonuclease and exonuclease activities and utilizes both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. Previously, studies of EBV DNase have been focused on the detection of anti-DNase antibodies in the patients with EBV associated diseases, the biochemical properties and the relationship between function and structure of the enzyme. In this dissertation, one monoclonal antibody (mAb) to EBV DNase was generated and the relationship of function and structure of EBV DNase including DNA binding domains and nuclear localization signals (NLSs) was further investigated and the effect of EBV DNase on the cell growth was examined. A mAb to EBV DNase, designated 311H, was obtained and the mAb can recognize the EBV DNase protein specifically. The antigenic epitope of 311H was located to a.a. 1-152 of EBV DNase. This mAb is useful for following studies of EBV DNase. To map regions of EBV DNase responsible for DNA binding activity, a series of mutant DNase polypeptides was expressed using an in vitro transcription/translation system and their DNA binding activities to DNA cellulose were determined. The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding. However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities. In the N-terminus, deletion of residues 23-28 and residues 7-61 re sulted in the loss of nuclease activity but the DNA binding activities of the trucated enzymes were intermediate and low, respectively. Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected. DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA. These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of intact and highly ordered structure(s) for both activities. It has been reported that EBV DNase may be detected in the nucleus and/or cytoplasm of infected cells. In this study, using cell-fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (-Gal). The results demonstr ated that two regions of the DNase with NLS activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the -Gal to the nuclei of HEp-2 cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS. In NLS-B, the histidine residue at amino acid 306 is required for NLS-B activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure. EBV DNase was expresed under inducible control of lactose operon to investigate its effects on cells. Induction of DNase expression with IPTG in Raj i cells caused DNA strand breaks, which were examined by using a comet assay. In addition, the expression of DNase resulted in decreased growth of Raji cells with cell cycle arrest at G1 and accumulation of cyclin E and p27Kip1. The results imply that EBV-DNase-induced DNA strand breaks may be associated with the chromosomal instability of cells. Chen Jen-Yang 陳振陽 1998 學位論文 ; thesis 121 en_US |