Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently.
Protein extracts were prepared at various times after serum stimulation of growth-arrested mouse 3T3 fibroblasts. The extracts were fractionated by sucrose gradient centrifugation and used to determine the activities of DNA polymerase tl and DNA primase. We found that polymerase and primase appeared in close association in one homogeneous 8.2-S peak. Neither polymerase, free of associated primase, nor primase, free of polymerase, could be detected at any time after serum stimulation. The activities of both enzymes started to increase concomitantly at the beginning of the DNA replication phase of the cell cycle. We found five to six times more DNA primase activity in replicating than in resting 3T3 cells.Besides DNA primase, a second additional priming activity could be detected. This activity sedimented at 12.5 S and corresponded most probably to RNA polymerase I.DNA primase initiates DNA synthesis by making short oligoribonucleotide primers, which are then elongated with DNA chains. Almost all nuclear mammalian DNA primases have been found in close association with DNA polymerase a. Isolated DNA polymerase tl consists of one large subunit of approximately 180 kDa and at least two smaller subunits of about 70 kDa and 55 kDa (recently reviewed in [l]). An apparently homogeneous DNA primase preparation, free of DNA polymerase, was isolated from mouse hybridoma cells. The hybridoma cell primase activity sedimented at 5.5 S and was associated with two polypeptides of 56 kDa and 46 kDa [2]. More recently a 5.5-S-primase-like activity could be recovered after treatment of purified mouse cell polymerase 01 with ethyleneglycol and dimethylsulfoxide [3, 41. The conclusion that DNA primase activity may be associated with one of the smaller polymerase subunits is consistent with reports describing a free DNA primase of 50-60 kDa from Drosophila melanogaster embryos [5] and yeast cells [6, 71. We would like to point out though that this conclusion has not remained unchallenged. Evidence has been presented suggesting that both functions, primase and polymerase, could be located on the large subunit of the enzyme [8,9]. In any case, since DNA primase is one of the key enzymes of the replication apparatus it is important to know how its activity is regulated. We have asked whether the enzymatic activity is expressed in a prereplicative phase or in the replicative phase (S phase) of the cell cycle. It has been well documented that the activity of DNA polymerase a increases with the beginning of the S phase (summarized in [l, 101); and recent evidence suggests that the activity increase is coupled to de novo synthesis of the large 180-kDa subunit. The biosynthesis of the smaller polymerase subunits is less strictly coupled to the S phase and could already be detected some time before the onset of DNA replication [lo].In the present work we have determined DNA primase and DNA polymerase activities at various times after mouse 3T3 fibroblasts had synchronously entered the cell cycle. OurCorrespondence to R. Knippers, Fakultat fur ...
We examine changes in protein synthesis that accompany suspension (i.e. shape alteration) of anchorage-dependent and anchorage-independent cells using a newly developed cell fractionation procedure based on detergent extraction. Using this procedure, a cell can be divided into four distinct and independent fractions: soluble, cytoskeleton, chromatin and nuclear matrix-intermediate filament. This fractionation procedure is used to investigate protein synthetic events associated with the release from anchorage-dependent growth, characteristic of transformed cells. Suspension results in several unexpected events in both anchorage-dependent (3T3) cells and anchorage-independent (SVPy 3T3) cells. Suspension of 3T3 cells results in a reduction of total protein synthesis; however, two proteins are enhanced in their amount of synthesis. Suspension of SVPy 3T3 results in about 20 proteins proceeding through short and long-term alterations in the rate of synthesis. The synthesis of some of these proteins is inhibited, others undergo a transient decrease in synthesis upon suspension and then increase above their rate in the anchored state, while the synthesis of others steadily increases after suspension. Suspension of anchorage-dependent cells results in a fraction specific shift in the rates of protein synthesis. Possible roles for these fraction-specific proteins are considered.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.