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Structure of a bifunctional DNA primase-polymerase
Georg Lipps1, Andreas O Weinzierl, Gudrun von Scheven
1Institute of Biochemistry, University of Bayreuth, Universitätsstrasse 30, D-95447 Bayreuth, Germany. georg.lipps@uni-bayreuth.de
Nature Structural & Molecular Biology
|January 20, 2004
Summary
Newly discovered archaeal replicases combine primase and DNA polymerase functions. Structural analysis reveals a unique mechanism, suggesting a common ancestor for archaeal and eukaryotic primases.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Genome replication typically involves separate primase and DNA polymerase enzymes.
- Novel replicases from archaeal plasmids, like pRN1, integrate both primase and DNA polymerase activities.
Purpose of the Study:
- To perform a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain.
- To elucidate the catalytic mechanism and evolutionary origins of this bifunctional enzyme.
Main Methods:
- X-ray crystallography to determine the 3D structure of the pRN1 prim-pol domain.
- Site-directed mutagenesis to investigate the roles of specific residues in DNA binding and catalysis.
- Biochemical assays to assess primase and DNA polymerase activities.
Main Results:
- The crystal structure revealed a central depression with conserved residues crucial for DNA binding and catalysis.
- Mutations affecting DNA affinity were identified on one side of the depression.
- Key acidic residues and a histidine on the opposite side are essential for both primase and polymerase activities, with one acidic residue coordinating a manganese ion.
- The structure shows no similarity to known DNA polymerases but is distantly related to archaeal and eukaryotic primases.
Conclusions:
- The pRN1 prim-pol domain possesses a unique catalytic mechanism, likely metal-dependent.
- The findings suggest a shared evolutionary origin between archaeal/eukaryotic primases and the pRN1 prim-pol domain, possibly stemming from a bifunctional replicase ancestral to small DNA genomes.