Association of poly(ADP-ribose) polymerase with the nuclear matrix: The role intermolecular disulfide bond formation, RNA retention, and cell type

Scott H Kaufmann, Genevieve Brunet, Brian Talbot, Daniel Lamarr, Carole Dumas, Joel H. Shaper, Guy Poirier

Research output: Contribution to journalArticle

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Abstract

The recovery of the enzyme poly(ADP-ribose) polymerase (pADPRp) in the nuclease- and 1.6 M NaCl-resistant nuclear subfraction prepared from a number of different sources was assessed by Western blotting. When rat liver nuclei were treated with DNase I and RNase A followed by 1.6 M NaCl, ~ 10% of the nuclear pADPRp was recovered in the sedimentable fraction. The proportion of pADPRp recovered with the residual fraction decreased to < 5% of the total nuclear polymerase when nuclei were prepared in the presence of the sulfhydryl blocking reagent iodoacetamide and increased to ~ 50% of the total nuclear pADPRp when nuclei were treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to fractionation. To determine whether this effect of disulfide bond formation was unique to rat liver nuclei, nuclear matrix/cytoskeleton structures were prepared in situ by sequentially treating monolayers of tissue culture cells with Nonidet-P40, DNase I and RNase A, and 1.6 M NaCl (S. H. Kaufmann and J. H. Shaper (1991) Exp. Cell Res. 192, 511-523). When nuclear monolayers were prepared from HTC rat hepatoma cells, CaLu-1 human lung carcinoma cells, and CHO hamster ovary cells in the absence of NaTT, pADPRp was undetectable in the nuclease- and 1.6 M NaCl-resistant fraction. In contrast, when nuclear monolayers were isolated in the presence of NaTT, from 5% (CaLu-1) to 26% (HTC cells) of the total nuclear pADPRp was recovered with the nuclease- and salt-resistant fraction. Examination of these residual structures by SDS-polyacrylamide gel electrophoresis under nonreducing conditions suggested that pADPRp was present as a component of disulfide cross-linked complexes. Further analysis by immunofluorescence revealed that the pADPRp was diffusely distributed throughout the CaLu-1 or CHO nuclear matrix. In addition, when matrices were prepared in the absence of RNase A, pADPRp was also observed in the residual nucleoli. These observations reveal that the recovery of pADPRp with a nuclease- and salt-resistant nuclear subfraction is dependent on the source of the nuclei and on the conditions used to fractionate those nuclei. In addition, these observations raise the possibility that there might be different functional classes of pADPRp molecules within the nucleus.

Original languageEnglish (US)
Pages (from-to)524-535
Number of pages12
JournalExperimental Cell Research
Volume192
Issue number2
DOIs
StatePublished - 1991
Externally publishedYes

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Nuclear Matrix
Poly(ADP-ribose) Polymerases
Disulfides
RNA
Pancreatic Ribonuclease
Sulfhydryl Reagents
Deoxyribonuclease I
Tetrathionic Acid
Salts
Cross-Linking Reagents
Iodoacetamide
CHO Cells
Liver
Cytoskeleton
Cricetinae
Fluorescent Antibody Technique
Polyacrylamide Gel Electrophoresis
Hepatocellular Carcinoma
Ovary
Cell Culture Techniques

ASJC Scopus subject areas

  • Cell Biology

Cite this

Association of poly(ADP-ribose) polymerase with the nuclear matrix : The role intermolecular disulfide bond formation, RNA retention, and cell type. / Kaufmann, Scott H; Brunet, Genevieve; Talbot, Brian; Lamarr, Daniel; Dumas, Carole; Shaper, Joel H.; Poirier, Guy.

In: Experimental Cell Research, Vol. 192, No. 2, 1991, p. 524-535.

Research output: Contribution to journalArticle

Kaufmann, Scott H ; Brunet, Genevieve ; Talbot, Brian ; Lamarr, Daniel ; Dumas, Carole ; Shaper, Joel H. ; Poirier, Guy. / Association of poly(ADP-ribose) polymerase with the nuclear matrix : The role intermolecular disulfide bond formation, RNA retention, and cell type. In: Experimental Cell Research. 1991 ; Vol. 192, No. 2. pp. 524-535.
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