Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

Abstract

DNA synthesis in a broken cellular preparation of Chinese hamster cells was enhanced approximately 10-fold by a brief trypsin treatment. Alphachywotrypsin also enhanced the, synthesis, whereas Pronase did not. The trypsin appears to be acting on a compopent of the nucleus. Evidence suggests that the trypsin is not removing protein from the DNA, but may be activating the system some other way.Much remains to be learned about DNA replication, especially in eukaryotic systems. The presence of histones, other chromosomal proteins, and the nuclear envelope provide extra complications for such studies 'in eukaryotes. Using cell culture systems, Taylor has shown that the nascent DNA is replicated in 6S units and then joined by a ligase (1). The same mechanism exists in prokaryotes (2). Recently it has been shown that a short chain of RNA is associated with the nascent DNA, and it has been suggested that this RNA must be synthesized before the DNA can be replicated (3). Silverman and Mirsky (4) have shown that exogenous DNA polymerase and RNA polymerase both have much less access to the DNA in isolated nuclei or chromatin than in isolated DNA.Using isolated nuclei, chromatin, and DNA, we have studied DNA synthesis using the endogenous DNA polymerase. In the course of such experiments we found a striking effect of trypsin on the rate of DNA synthesis. Suspecting at first that the effect might be due to the removal of histones from the DNA, we were surprised to find that this was not the explanation. The key experiments will be reported in this paper; a preliminary abstract was published earlier (5). MATERIALS AND METHODSCulture&. Chinese hamster fibroblast cultures (Don and Don-C) were grown as monolayers in McCoy's 5a medium supplemented with 10% fetal-calf serum (v/v).I8olation of Nuclei and(Cytosol&. Nuclei were isolated by a modification of the method of Wray and Stubblefield (6). The isolation buffer contained 1 mM CaCl2, 0.5 mM piperazine-NN'-bis(2-ethanesulfonic acid) monosodium monohydrate "PIPES" (Calbiochem) at pH 6.7, and 0.5 M hexylene glycol. Cells were either removed by a brief trypsin treatment or else scraped off in isolation buffer with a rubber policeman. In either case, the cells were washed once in 4 ml of isolation buffer (5 X 106 cells per ml) at 40 and then pelleted by centrifugation at 800 X g for 5 min at 4°. The pellet was then resuspended in isolation buffer at approximately 2 X 107 cells per ml. The nuclei were isolated by rapid 2432 passage of the suspension through a 22-gauge needle with the bevel placed against the side of the centrifuge tube. This was repeated until clean nuclei were obtained, as determined by phase microscopy. The resulting preparation is called the cellular lysate. The nuclei can be removed from the suspension by centrifugation at 800 X g for 5 min at 4°. In some cases the supernatant from this was' then centrifuged again at 100,000 X g at 4°. The supernatant from the high-speed centrifugation is termed the cytoplasmic cyto8ol. The pellet of nuclei was resuspende...

Influence of adeno-associated virus on adherence and growth properties of normal cells

“…Influence of cell detachment on resumption of cell proliferation. It has been described that trypsin treatment stimulates DNA synthesis in cell-free systems (8,9) and induces DNA synthesis and cell proliferation in contact-inhibited fibroblast cultures (10,24,42). Furthermore, it is known that loosening the attachment of cells to their substratum is required for proliferation (10,24,42).…”

Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system

“…Although these effects of trypsin might be expected to influence the capacity of isolated nuclei to synthesize DNA, we did not observe any differences when comparing them with nuclei isolated from cells scraped from glass surfaces. Brown & Stubblefield (1974) also found that DNA synthesis was stimulated about 10-fold in Chinesehamster fibroblast-cell lysates treated with trypsin. DNA synthesis in our BHK-cell nuclei was inhibited by 90 % after exposure to trypsin.…”