Preferential binding of DNA primase to the nuclear matrix in HeLa cells.
Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.
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Collins
D000255:Adenosine Triphosphate / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology*; D004257:DNA Polymerase II / Q000032:analysis / Q000302:isolation & purification; D019915:DNA Primase; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D011071:Poly T / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000032:analysis* / Q000302:isolation & purification; D012995:Solubility
D011071:Poly T; D000255:Adenosine Triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II