Cell-cycle-dependent expression of DNA primase activity

Abstract

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 ...

Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

“…Measurements of enzyme activity showed that after serum stimulation, primase activity increased about five-to sixfold and that this increase occurred along with the induction of DNA synthesis (25), consistent with our observation that mRNA expression preceded DNA synthesis. However, there appeared to be several hours before DNA synthesis during which p49 mRNA was present before primase activity was detected.…”

Maize DNA Polymerase 2 is a Phosphoprotein with Increasing Activity During Germination

“…The relationship between DNA replication and DNA polymerase u activity has been studied under different conditions of cell growth and differentiation (Thommes et al, 1986;Reiter et al, 1987;Cripps-Wolfman et al, 1989;Nasheuer et al, 1991). Results have shown that changes in DNA polymerase a activity take place during the cell cycle with an increase in activity prior to the start of the S phase (Baril et al, 1973;Spadari and Weissbach, 1974;Chiu and Baril, 1975;Bertazzoni et al, 1976;Tanabe et al, 1984;Cripps-Wolfman et al, 1989).…”