Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver.

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal human mesothelial cells and fibroblasts transfected with the EJras oncogene become EGF-independent, but are not malignantly transformed.

The nature of the lesion in growth control exerted by the cancer-derived c-H-ras mutation, EJ-ras, and its transforming potential in diploid cells are both poorly understood. We introduced EJ-ras into normal, diploid human mesothelial cells and fibroblasts and obtained transfectants expressing p21EJ-ras. All clones examined were independent of EGF for rapid growth, and all secreted an EGF-like mitogen into the medium at levels sufficient to satisfy the EGF requirement of normal cells. The EJ-ras transfectants were not altered with respect to any other growth requirement, and they were not transformed. Eleven clones tested all retained a finite replicative lifespan which, in most cases, was the same as that of the parent cell strain. Three transfectants tested were not tumorigenic in nude mice. Thus p21EJ-ras can circumvent an important mitogenic signal pathway in human cells. Nevertheless, neither the secretion of an autocrine growth factor nor any other effect of p21EJ-ras serves to malignantly transform normal human cells, in contrast to the susceptibility of some established rodent cell lines to transformation by these mechanisms.