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Preferential binding of DNA primase to the nuclear matrix in HeLa cells
Abstract:
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.
Insights
This study detects DNA primase bound to the HeLa nuclear matrix, showing most primase activity is matrix-bound, unlike DNA polymerase alpha. This supports the nuclear matrix
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The nuclear matrix plays a crucial role in the spatial organization of DNA replication.
- Previous work established a link between DNA synthesis rates and DNA polymerase alpha binding to the nuclear matrix during S-phase.
Purpose of the Study:
- To detect and characterize DNA primase bound to the HeLa nuclear matrix.
- To investigate the subcellular distribution and matrix association of DNA primase and DNA polymerase alpha.
Main Methods:
- Detection of matrix-bound DNA primase activity using [32P]dAMP incorporation into poly(dT) and [3H]AMP incorporation into matrix DNA.
- Assay of primase and polymerase alpha activity in subcellular fractions and after solubilization with beta-D-octylglucoside.
- Characterization of primase activity using specific inhibitors like adenosine 5'-O-(thiotriphosphate).
Main Results:
- DNA primase was detected bound to the HeLa nuclear matrix.
- Approximately 72% of total primase activity was matrix-bound, compared to only 32% of DNA polymerase alpha activity.
- Beta-D-octylglucoside solubilized a significant portion of both matrix-bound primase (54%) and polymerase alpha (39%).
Conclusions:
- The findings provide further evidence for a structural and functional role of the nuclear matrix in DNA replication.
- The differential matrix association of primase and polymerase alpha suggests distinct roles in replication organization.
- Solubilization of matrix-bound enzymes offers a method for studying their function in DNA replication spatial organization.