|Budget Amount *help
¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥5,400,000 (Direct Cost : ¥5,400,000)
Previously, we showed that DNA polymerase alpha, beta, and gamma were strongly supressed by PI, which bound to hydrophobic region (DNA binking domain) of the enzyme molecules (Yoshida et al. 1988). It was also shown that PI and cardiolipin strongly inhibited the in vitro replication of human c-myc ARS. This inhibition is due, at least in part, to the inhibition of topoiso merase I (Umekawa et al.). It was reported that PIP or IP2 activated DNA polymerase alpha (Syl via et al.). These results suggest that DNA replication is regulated by PI and its metabolites. In this context, we tested the effects of phosphoinositides or their derivatives and found that a crude fraction of DNA polymerase alpha (low specific activity form) was stimulated by IP2. However, After purification of the fraction by Mono Q column chromatography (FPLC, Phrmiacia), the stimulation disappeared, and was restored by the addition of flow-through fraction. These results indicate that IP2 stimulate enzyme indirectly, via an modifing factor of DNA polymerase alpha. We are now purifing this protein factor. On the other hand, we also found that cell nuclei contained a considerable amount of phospholipase C (PL C) which is specific for phosphoinositides. The nuclear PLC was purified and separated into four distinct molecular species. One of them, 88 kDa nPLC, was found to be specific to nuclei of growing cells, such as regenerating liver or ascites hepatoma, but absent in resting liver cell. This activity increased transiently at S-phase of liver regeneration.