The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis.
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.
Institute of Biochemistry, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany.
Kirsten
K
Beck
Alessandro
A
Vannini
Patrick
P
Cramer
Georg
G
Lipps
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000096:biosynthesis*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013698:Templates, Genetic
D017931:DNA Primers; D019915:DNA Primase