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Stimulation of DNA polymerase alpha activity by microtubule-associated proteins
M Shioda1, K Okuhara, H Murofushi
1Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly 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. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.
Insights
Microtubule-associated protein 2 (MAP2) enhances DNA polymerase alpha activity by binding to it, increasing DNA synthesis initiation. This specific stimulation suggests MAPs may play a role in DNA replication.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Microtubule-associated proteins (MAPs) are crucial for microtubule dynamics.
- DNA polymerases are essential enzymes for DNA replication and repair.
- The interaction between MAPs and DNA polymerases is not well understood.
Purpose of the Study:
- To investigate the effect of Microtubule-associated protein 2 (MAP2) on DNA polymerase alpha activity.
- To determine the mechanism by which MAP2 influences DNA polymerase alpha.
- To explore the specificity of MAP2's interaction with different DNA polymerases.
Main Methods:
- Purification of DNA polymerase alpha from calf thymus and human lymphoma cells.
- Isolation of MAP2 from porcine brains.
- Enzyme activity assays using various DNA templates and primers.
- Kinetic analysis (Km and Vmax determination).
- Affinity chromatography using MAP2-immobilized columns.
Main Results:
- MAP2 dose-dependently stimulated DNA polymerase alpha activity, particularly with activated DNA or poly(dA).(dT)10 templates.
- Physical binding between DNA polymerase alpha and MAP2 was demonstrated.
- MAP2 decreased the Km for deoxyribonucleotides and template-primers, while increasing Vmax, suggesting enhanced initiation frequency.
- MAP2 specifically stimulated DNA polymerase alpha, not other DNA polymerases (beta, gamma, E. coli I).
- Other MAPs (tau, 190-kDa MAP) could also substitute for MAP2.
Conclusions:
- MAP2 physically binds to and specifically stimulates DNA polymerase alpha activity.
- The stimulation mechanism involves increased initiation frequency of DNA synthesis.
- These findings suggest a potential role for MAPs in DNA replication in vivo.