Deoxyribonuclease activity found in Epstein-Barr virus producing lymphoblastoid cells

Clough, Wendy

doi:10.1021/bi00588a009

Cited by 44 publications

(49 citation statements)

References 19 publications

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“…We have shown that the DNase is separable from the polymerase after several stages of purification and that it digests both native and denatured DNA, used as substrate, to mononucleotides (2). We report here that this EBV-induced exonuclease is inseparable through multiple purification steps from an endonucleolytic activity that also is EBV-induced and acts in a selective manner on phage X DNA used as substrate.…”

mentioning

confidence: 85%

“…2 bears a strong resemblance to the recBC gene products isolated from E. coli. This prokaryotic nuclease has been shown to contain endonucleolytic and single-and double-stranded exonucleolytic activities that are not separable after extensive purification (17).…”

Section: Microbiology: Cloughmentioning

confidence: 96%

“…The HR-I cell line, originally isolated from a Burkitt tumor, was used for the enzyme preparations shown here. Two DEAE-cellulose column chromatography steps were followed by phosphocellulose chromatography (1,2) and, in the case of the exonuclease, by native DNA-cellulose chromatography (2). the digested X DNA (Fig.…”

Section: Copurification Of Exonucleolytic and Endonucleolyticmentioning

confidence: 99%

“…However, it was shown in separate experiments that phosphocellulose-purified exonuclease, when sedimented in glycerol gradients under high-salt conditions, also contained marked endonuclease activity, indicating that glycerol sedimentation under nonaggregating conditions was also not sufficient to separate these two nuclease activities (ref. 2 …”

Section: Copurification Of Exonucleolytic and Endonucleolyticmentioning

confidence: 99%

“…While recBC has been studied as an important component in the process of DNA recombination, it in fact may also be involved in the process of DNA replication (20), a role postulated for the EBV-induced nuclease activities as well (ref. 2 complexed with DNA polymerase (20), as are the EBV-induced nucleases (2). An important difference between the prokaryotic and the eukaryotic nucleases is that recBC also contains an active DNA-dependent ATPase and the nuclease has a strong ATP requirement (18).…”

Section: Microbiology: Cloughmentioning

confidence: 99%

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An endonuclease isolated from Epstein-Barr virus-producing human lymphoblastoid cells.

Clough¹

1980

Proc. Natl. Acad. Sci. U.S.A.

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An endonuclease has been isolated from human B lymphoblastoid cells that copurifies with an exonucleolytic activity and has been shown to produce double-strand breaks and a high proportion of single-strandedness in phage X DNA in vitro. The data are consistent with a model in which single-strand cuts are made by the endonucleolytic activity, possibly in A+T-rich regions of the DNA, followed by creation of single-stranded regions (gaps) precessing from the site of a cut. Generation of overlapping gaps on opposite strands or of a gap opposite a nick would lead to the creation of the banding patterns that we have seen on electrophoretic gels. This endonucleolytic activity copurifies with other enzymes induced by Epstein-Barr virus that relate to the process of viral DNA replication in productively infected cells. However, a more general role is proposed for this class of eukaryotic endonuclease activities. A marked degree of single-strandedness has been found in the replicating DNAs of many eukaryotes, and these gaps could be generated by endonucleases with associated exonucleolytic activity such as that reported here. This EpsteinBarr virus-induced nuclease activity has been shown to resemble the recBC nuclease isolated from the prokaryote Escherichia coli and also the endonuclease isolated from the eukaryote Chlamydomonas.Our laboratory has recently reported that human B lymphoblastoid cell lines that are productively infected with Epstein-Barr virus (EBV) contain EBV-induced DNA polymerase (1) and DNase (2) activities. We have shown that the DNase is separable from the polymerase after several stages of purification and that it digests both native and denatured DNA, used as substrate, to mononucleotides (2). We report here that this EBV-induced exonuclease is inseparable through multiple purification steps from an endonucleolytic activity that also is EBV-induced and acts in a selective manner on phage X DNA used as substrate. This EBV-induced endonuclease reduces X DNA to a discontinuous series of large fragments that are then very sensitive to the action of S1 endonuclease. However, DNA fragments of similar length, generated from the large X genome, result from this endonucleolytic reaction only in the presence of concentrations of salt sufficient to inhibit the active exonucleolytic activity (160-180 mM KCI).The EBV-induced endonuclease reported here resembles in many important respects an enzymatic activity isolated from the eukaryote Chlamydomonas by Burton et al. (3). The Chlamydomonas endonuclease has been postulated to cut at specific sites on one strand and to then digest one of the two strands of the substrate DNA. The gel electrophoretic pattern of fragments generated by treatment of DNA with various concentrations of the Chlamydomas enzyme resembles the pattern of digests shown in the present report that result from the action of the EBV-induced endonuclease on phage X DNA.We also suggest that this class of eukaryotic nuclease resembles the prokaryotic nuclease recBC isolated from Escherichi...

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mentioning

confidence: 85%

Section: Microbiology: Cloughmentioning

confidence: 96%

Section: Copurification Of Exonucleolytic and Endonucleolyticmentioning

confidence: 99%

Section: Copurification Of Exonucleolytic and Endonucleolyticmentioning

confidence: 99%

Section: Microbiology: Cloughmentioning

confidence: 99%

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An endonuclease isolated from Epstein-Barr virus-producing human lymphoblastoid cells.

Clough¹

1980

Proc. Natl. Acad. Sci. U.S.A.

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Immunological characterization of the epstein‐barr virus phosphoprotein pp58 and deoxyribonuclease expressed in the baculovirus expression system

Chen¹,

Sauter²,

Haiss³

et al. 1991

Intl Journal of Cancer

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The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

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Identification of an Epstein-Barr virus-coded thymidine kinase.

Littler¹,

Zeuthen²,

McBride³

et al. 1986

The EMBO Journal

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We have demonstrated the presence of an Epstein‐Barr virus (EBV)‐coded thymidine kinase (TK) by producing biochemically transformed, TK‐positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate‐mediated transfection of the SalI‐B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI‐B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross‐reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK‐ strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV‐1 revealed significant regions of homology.

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Deoxyribonuclease activity found in Epstein-Barr virus producing lymphoblastoid cells

Cited by 44 publications

References 19 publications

An endonuclease isolated from Epstein-Barr virus-producing human lymphoblastoid cells.

An endonuclease isolated from Epstein-Barr virus-producing human lymphoblastoid cells.

Immunological characterization of the epstein‐barr virus phosphoprotein pp58 and deoxyribonuclease expressed in the baculovirus expression system

Identification of an Epstein-Barr virus-coded thymidine kinase.

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