Crystal structure of a DNA-dependent RNA polymerase (DNA primase)

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 Å resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.

[1]  A. D. Clark,et al.  Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[3]  W. Copeland,et al.  Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. , 1993, The Journal of biological chemistry.

[4]  F. Grosse,et al.  DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity. , 1988, The Journal of biological chemistry.

[5]  T. Kusakabe,et al.  Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-oligonucleotides at Primase Recognition Sites* , 1997, The Journal of Biological Chemistry.

[6]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[7]  W. Copeland,et al.  Active Site Mapping of the Catalytic Mouse Primase Subunit by Alanine Scanning Mutagenesis (*) , 1995, The Journal of Biological Chemistry.

[8]  R. Conaway,et al.  A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Detlef D. Leipe,et al.  Did DNA replication evolve twice independently? , 1999, Nucleic acids research.

[10]  R. Kuchta,et al.  Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology. , 1999, Biochemistry.

[11]  D. Wigley,et al.  Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase. , 2000, Structure.

[12]  K. Singh,et al.  A unified DNA- and dNTP-binding mode for DNA polymerases. , 1998, Trends in biochemical sciences.

[13]  R. W. Smith,et al.  Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. , 1998, The Journal of biological chemistry.

[14]  Samuel H. Wilson,et al.  Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. , 1997, Biochemistry.

[15]  J. Ito,et al.  Compilation, alignment, and phylogenetic relationships of DNA polymerases. , 1993, Nucleic acids research.

[16]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[17]  Detlef D. Leipe,et al.  Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. , 1998, Nucleic acids research.

[18]  P. Brick,et al.  Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii. , 1999, Nucleic acids research.

[19]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[20]  Thomas A. Steitz,et al.  Structure of Taq polymerase with DNA at the polymerase active site , 1996, Nature.

[21]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.

[22]  J. Kuriyan,et al.  A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases. , 2000, Journal of molecular biology.

[23]  R. Read,et al.  Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Steitz,et al.  A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. , 1994, Science.

[25]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[26]  P. Borer,et al.  Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. , 1998, Science.

[27]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[28]  G. Lucchini,et al.  The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis. , 1993, The Journal of biological chemistry.

[29]  T. Kusakabe,et al.  The Role of the Zinc Motif in Sequence Recognition by DNA Primases* , 1996, The Journal of Biological Chemistry.

[30]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[31]  J. Berger,et al.  Structure of the RNA polymerase domain of E. coli primase. , 2000, Science.