The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the of human DNA polymerase {alpha}-primase complex [Enzymology]

June 25th, 2014 by Zhang, Y., Baranovskiy, A. G., Tahirov, T. H., Pavlov, Y. I.

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase (polα-prim)4 complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pull-down assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, while the whole catalytically active heterodimer of polα (p180∆N-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180∆N-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180∆N-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70 and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homo-pyrimidine runs create a strong barrier for DNA synthesis by polα.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the of human DNA polymerase {alpha}-primase complex [Enzymology]