DNA Synthesis by The Isolated Nuclear Matrix and Its Regulatory Mechanism

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The nuclear matrix plays a role as the site of DNA replication in eukaryotic cells. It was found that multi-enzyme complexes including DNA polymerase alpha, one of the replicative enzymes of eukaryotes, were associated with the matrix, and the matrix isolated from a true slime mold, Physarum polycephalum, was able to catalyze the formation of replication intermediates in vitro. Similar stimulation occurred in the nuclear matrices isolated from HeLa cells and rat ascites hepatoma cells. DNA polymerase alpha bound to a MAP-immobilized column, whereas preincubation of the enzyme with unbound MAPs prevented binding to the column. Kinetic analyzes revealed that MAPs decreased the Km value of the polymerase for deoxyribonucleoside triphosphates, irrespective of the species of template-primer. Product analyzes showed that MAPs did not alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. These findings indicate that a putative DNA replication machinery including DNA polymerase alpha exists on the matrix.MAPs, which are the general term for the accessory proteins associated with microtubules, one of the major cytoskeletal components, stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. MAPs also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. MAPs stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Kinetic analyzes showed that MAPs decreases the Km value of the matrix for deoxyribonucleoside triphosphates. Comparison of the Km values of active- and latent-type DNA replication machineries of Physarum suggested a possible role for MAPs or MAP-like proteins in DNA replication.