Abstract
The B subunit of the DNA polymerase (pol) alpha-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180.p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180.p86 subcomplex is required for B subunit phosphorylation.
Highlights
The development of cell-free systems capable to replicate viral DNA molecules has been essential in understanding the fundamental enzymology of eukaryotic DNA replication and in identifying most of the required cellular factors (Challberg and Kelly, 1989; Stillman, 1989; Hurwitz et al, 1990; Waga and Stillman, 1994)
The fully assembled pol ␣-primase complex found in ␣-factor arrested cells (Time 0 in Fig. 1) has to be inherited from the previous cell cycle, since it is known that the pol ␣-primase polypeptides are periodically synthesized, following the transient transcription of their corresponding genes in G1/S (Foiani et al, 1995; Johnston et al, 1990; Foiani et al, 1989; Johnston et al, 1987)
Maternal p86 is phosphorylated early in S phase, while the newly synthesized p86, which derives from the periodic transcription of the corresponding POL12 gene, is phosphorylated in late S/early G2 phase 70 min after its synthesis, and p91 is dephosphorylated to p86, while cells are exiting from mitosis (Foiani et al, 1995)
Summary
The development of cell-free systems capable to replicate viral DNA molecules has been essential in understanding the fundamental enzymology of eukaryotic DNA replication and in identifying most of the required cellular factors (Challberg and Kelly, 1989; Stillman, 1989; Hurwitz et al, 1990; Waga and Stillman, 1994). We show that the four subunits of the yeast DNA polymerase ␣-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol ␣ (p180) and unphosphorylated B subunit (p86) occurs very rapidly.
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