Temperature-dependent association of DNA polymerase alpha activity with the nuclear matrix.

Abstract:

We have investigated the effect of preincubating isolated nuclei at the physiological temperature of 37 degrees C on the recovery of DNA polymerase alpha and beta activities bound to the nuclear matrix. In HeLa cells, when purified nuclei are incubated for at least 30 min at 37 degrees C prior to extraction with 2 M NaCl and digestion with DNase I, about 30% of nuclear DNA polymerase alpha activity is associated with the final matrix along with about 20% of nuclear protein. If the preincubation is carried out at 0 degrees C, less than 5% of the enzyme activity is resistant to high salt extraction and the protein recovery drops to about 12%. On the contrary, the recovery of nuclear DNA polymerase beta activity bound to the matrix fraction is independent of the temperature at which the preincubation is performed. The same levels of DNA polymerase alpha activity are found to be matrix associated even if reducing and chelating agents are present during the exposure of isolated nuclei to 37 degrees C, suggesting that this phenomenon does not depend on the in vitro formation of disulfide bonds or on some metal ion-protein interaction. Our data could explain why, in the past, different results have been obtained when the association of DNA polymerase alpha with the nuclear matrix has been analyzed.

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