The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain 
for faithful primer synthesis

Beck, K.; Vannini, A.; Cramer, P.; Lipps, G.

doi:10.1093/nar/gkq447

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The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis

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Cramer, P.
Department of Molecular Biology, MPI for Biophysical Chemistry, Max Planck Society;

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Citation

Beck, K., Vannini, A., Cramer, P., & Lipps, G. (2010). The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis. Nucleic Acids Research, 38(19), 6707-6718. doi:10.1093/nar/gkq447.

Cite as: https://hdl.handle.net/11858/00-001M-0000-0015-8226-3

Abstract

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40–370. While the N-terminal part of that minimal region (residues 47–247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248–370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.