No discrete complexes containing DNA polymerase alpha activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells.
Most of the DNA polymerase alpha activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5-20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase alpha activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37 degrees C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase alpha to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.
Martelli
A M
AM
Dipartimento di Morfologia Umana Normale, Università di Trieste, Italy.
Cocco
L
L
D002499:Centrifugation, Density Gradient; D004257:DNA Polymerase II / Q000378:metabolism*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D015530:Nuclear Matrix / Q000201:enzymology*; D012995:Solubility
D004257:DNA Polymerase II