Highly efficient copying of single-stranded DNA by eukaryotic cell chromatin

Abstract

Chromatin prepared from S phase hepatoma tissue culture (HTC) cell incorporates in vitro about 11-14 pmoles [3H]dTMP into DNA in 30 min. Single-stranded DNA added to this chromatin stimulates DNA synthesis more than 40-fold whereas activated DNA enhances it about 60-fold. By contrast, stimulation of DNA synthesis by activated DNA in a crude nuclear extract exceeds the stimulation exerted by denatured DNA by a factor of 7. Stimulation of DNA synthesis by denatured DNA is not due to stabilization of either the chromatin or the product of the endogenous reaction. On the other hand, we find that poly(dC) and poly (dT) enhance DNA synthesis by serving as templates which are copied by chromatin in a true complementary fashion. It seems therefore, that eukaryotic cell chromatin is able to copy single-stranded DNA at a high efficiency. Chromatin of G1 arrested cell copies exogenous templates at a considerably reduced rate. The enzyme responsible for the copying of denatured DNA is tentatively identified as DNA polymerase alpha on the basis of its sensitivity to sulfhydril group blocking, its requirements for ions and failure to copy the ribo strand of oligo(dT) poly(A).

On the activity and fidelity of chromatin‐associated hepatic DNA polymerase‐β in aging murine species of different life spans

“…Since accuracy of DNA synthesis by any polymerase may be altered during purification of the enzyme, we have chosen to use crude polymerase preparations, which may better reflect the in vivo accuracy of the enzyme. Isolated chromatin which is capable of copying exogenous DNA templates retains some properties of the in vivo DNA replicating machinery (Kaftory and Fry, 1978;Wiesman-Shomer et al, 1979). In addition, distinct polymerase types associated with chromatin can be discerned by specific inhibitors and templates (Kaftory and Fry, 1978;Fry et al, 1980).…”

“…Isolated chromatin which is capable of copying exogenous DNA templates retains some properties of the in vivo DNA replicating machinery (Kaftory and Fry, 1978;Wiesman-Shomer et al, 1979). In addition, distinct polymerase types associated with chromatin can be discerned by specific inhibitors and templates (Kaftory and Fry, 1978;Fry et al, 1980). We find that DNA polymerase-@ is the major detectable polymerase species that associates with chromatin from mature mouse liver (Table 2).…”

“…The reaction was terminated either by adding 20-fold excess of unlabeled deoxyribonucleoside triphosphates or by introduction of hexokinase and glucose (Kaftory and Fry, 1978). Chromatin DNA was subsequently digested by a series of restriction endonucleases incapable of cleaving poly [d(A-TI].…”

“…(50-150 dpdpmol) and [3H]-dGTP (4-6 x lo* dpdpmol) was conducted for 2 hours in a DNA synthesis reaction mixture which also contained 400 pM actinomycin D in a final volume of 250 pl. The reaction was terminated either by adding 20-fold excess of unlabeled deoxyribonucleoside triphosphates or by introduction of hexokinase and glucose (Kaftory and Fry, 1978). Chromatin DNA was subsequently digested by a series of restriction endonucleases incapable of cleaving poly [d(A-TI].…”

“…musculus and P . leucopus Chromatin prepared from a variety of eucaryotic cells is capable of both endogenous DNA synthesis (Knopf and Weissbach, 1977;Kaftory and Fry, 1978) and of copying numerous exogenous DNA templates (Kaftory and Fry, 1978;Weisman-Shomer et al, 1979). The efficiencies of the endogenous reaction and of copying exogenous templates in chromatin isolated from livers of young and old M .…”