UBC Theses and Dissertations
UBC Theses and Dissertations
DNA polymerases in developing rat brain Chiu, Jen-fu
DNA polymerase activity in soluble extracts from developing rat brain described here shows many similarities to other animal DNA polymerases. Two DNA polymerases, A and B, have been separated and purified from 10-day-old rat brain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose. Some properties of these two enzymes differed considerably. Enzyme A was 2-3 times more active with heat-denatured DNA than with native DNA as template and Enzyme B almost always used native DNA as template. The optimal concentration of Mn²⁺ was 0.05 mM for Enzyme A and 0.3 mM for Enzyme B. ATP stimulated only Enzyme A, but EDTA stimulated Enzyme B and showed little or no effect on Enzyme A. Enzyme B was strongly stimulated by KCl. In the case of Enzyme A, however, salt gave no activation but rather a marked inhibition. Enzyme A was more sensitive to dithiothreitol and sulfhydryl-blocking agents than Enzyme B. The incorporation was linearly proportional to the Enzyme A concentration, but Enzyme B showed a sigmoidal slope in its enzyme concentration curve. Enzyme A sediments at around 9 S on sucrose gradients and Enzyme B at around 3-4 S. The pattern of developmental changes in two DNA polymerases of rat brain was studied. In the cerebellum, DNA polymerase A is the more active in very young animals but peaks at around the 6th day after birth. DNA polymerase B is more active than Enzyme A in the cerebella of older animals. In the cerebral cortex DNA polymerase A activity is higher than that of Enzyme B in the fetal stage but the activity of polymerase A is much lower than that of polymerase B at all post-natal ages studied. An extremely high activity of DNA polymerase B was solubilized from the nuclear membrane-chromatin complex isolated from rat brain. The relative amounts of soluble and particulate forms of DNA polymerase in the cerebella of rats change with age. Much of the activity is in the soluble form in younger rats, but in the adult rat, the DNA polymerase exists almost exclusively in a particulate form which is inactive unless solubilized as described. In younger rat brain, DNA polymerase A only exists in the soluble form and is localized both in the nucleoplasm and cytoplasm fraction. However, there is no, or very little, DNA polymerase A activity in adult rat brain. DNA polymerase B exists in both the soluble and particulate form in younger rat brain. The soluble form of DNA polymerase B is localized in the cytoplasm and nucleoplasm. The major part of DNA polymerase B exists as the particulate form in the nuclei at older stages. A protein factor was isolated from DNA polymerase B. The factor stimulated DNA polymerase B activity with double stranded DNA as template and showed no effect on DNA synthesis with single stranded DNA as template. It seems that this factor is specific for DNA polymerase B. The stimulatory effect was not due to the activity of a nuclease or nickase. The polyamines, i.e. spermine, spermidine and putrescine, enhanced the DNA polymerase activity in extracts of brain from 10-day-old rats by more than 50% and stimulated purified DNA polymerase B about 100%. However, polyamines showed little or no effect on DNA polymerase A activity. The mechanism of spermidine stimulation of DNA polymerase B activity is presumably through a facilitation or stabilization of the association of enzyme and the protein factor.
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