The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis |
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| Authors: | Kirsten Beck Alessandro Vannini Patrick Cramer Georg Lipps |
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| Affiliation: | 1.Institute of Biochemistry, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, 2.Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich CIPSM, Ludwig-Maximilians-Universität München, Feodor-Lynen-strasse 25, 81377 Munich, Germany and 3.University of Applied Research of Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Switzerland |
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| 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. |
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