Crystal Structure of the Helicase Domain from the Replicative Helicase-Primase of Bacteriophage T7

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.

[1]  C. Richardson,et al.  The Acidic Carboxyl Terminus of the Bacteriophage T7 Gene 4 Helicase/Primase Interacts with T7 DNA Polymerase* , 1997, The Journal of Biological Chemistry.

[2]  B. Oh,et al.  Crystal Structure of RNA Helicase from Genotype 1b Hepatitis C Virus , 1998, The Journal of Biological Chemistry.

[3]  J. Steitz,et al.  Alive with DEAD proteins , 1991, Nature.

[4]  A. Kwong,et al.  Structure of the hepatitis C virus RNA helicase domain , 1997, Nature Structural Biology.

[5]  D E Tronrud,et al.  TNT refinement package. , 1997, Methods in enzymology.

[6]  J. Griffith,et al.  Formation of a DNA Loop at the Replication Fork Generated by Bacteriophage T7 Replication Proteins* , 1998, The Journal of Biological Chemistry.

[7]  Tania A Baker,et al.  Polymerases and the Replisome: Machines within Machines , 1998, Cell.

[8]  Gabriel Waksman,et al.  Major Domain Swiveling Revealed by the Crystal Structures of Complexes of E. coli Rep Helicase Bound to Single-Stranded DNA and ADP , 1997, Cell.

[9]  M. Hingorani,et al.  The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Egelman,et al.  Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Griffith,et al.  A Domain of the Gene 4 Helicase/Primase of Bacteriophage T7 Required for the Formation of an Active Hexamer (*) , 1995, The Journal of Biological Chemistry.

[12]  S V Evans,et al.  SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. , 1993, Journal of molecular graphics.

[13]  F. Studier,et al.  Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. , 1992, The Journal of biological chemistry.

[14]  L. Romano,et al.  Nucleotide and DNA-induced Conformational Changes in the Bacteriophage T7 Gene 4 Protein (*) , 1995, The Journal of Biological Chemistry.

[15]  G. Waksman,et al.  Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super‐families of helicases , 1998, Protein science : a publication of the Protein Society.

[16]  N. Chandra,et al.  STRUCTURE OF RECA PROTEIN , 2000 .

[17]  R. Vale,et al.  Switches, latches, and amplifiers: common themes of G proteins and molecular motors , 1996, The Journal of cell biology.

[18]  J P Griffith,et al.  Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. , 1998, Structure.

[19]  Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs. , 1998, Nucleic acids research.

[20]  J. Griffith,et al.  Coordinated leading and lagging strand DNA synthesis on a minicircular template. , 1998, Molecular cell.

[21]  F. Studier,et al.  Biochemical Analysis of Mutant T7 Primase/Helicase Proteins Defective in DNA Binding, Nucleotide Hydrolysis, and the Coupling of Hydrolysis with DNA Unwinding* , 1996, The Journal of Biological Chemistry.

[22]  M. Hingorani,et al.  Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins. , 1993, The Journal of biological chemistry.

[23]  S. W. Matson,et al.  DNA helicases: Enzymes with essential roles in all aspects of DNA metabolism , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[24]  D Eisenberg,et al.  3D domain swapping: A mechanism for oligomer assembly , 1995, Protein science : a publication of the Protein Society.

[25]  W. Kabsch,et al.  The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. , 1997, Science.

[26]  M. Hingorani,et al.  Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases. , 1996, Biochemistry.

[27]  Jan Pieter Abrahams,et al.  Structure at 2.8 Â resolution of F1-ATPase from bovine heart mitochondria , 1994, Nature.

[28]  K. Bjornson,et al.  Mechanisms of helicase-catalyzed DNA unwinding. , 1996, Annual review of biochemistry.

[29]  E. Egelman.  Homomorphous hexameric helicases: tales from the ring cycle. , 1996, Structure.

[30]  L. Bird,et al.  Helicases: a unifying structural theme? , 1998, Current opinion in structural biology.

[31]  G. Schulz.  Binding of nucleotides by proteins , 1992, Current Biology.

[32]  L. Bird,et al.  Crystal structure of a DExx box DNA helicase , 1996, Nature.

[33]  T. Steitz,et al.  Structure of the recA protein–ADP complex , 1992, Nature.

[34]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[35]  S. Velankar,et al.  Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism , 1999, Cell.

[36]  K. Johnson,et al.  A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. , 1997, Biochemistry.

[37]  F. Studier,et al.  Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase. , 1992, The Journal of biological chemistry.

[38]  C. Richardson,et al.  DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7. , 1983, The Journal of biological chemistry.

[39]  C. Richardson,et al.  The gene 4 protein of bacteriophage T7. Characterization of helicase activity. , 1983, The Journal of biological chemistry.

[40]  A. Wittinghofer.  Signal transduction via Ras. , 1998, Biological chemistry.

[41]  J. Steitz,et al.  RNA splicing. Alive with DEAD proteins. , 1991, Nature.

[42]  K. Baradaran,et al.  An N-terminal fragment of the gene 4 helicase/primase of bacteriophage T7 retains primase activity in the absence of helicase activity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

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

[44]  M. Hingorani,et al.  The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation. , 1994, Biochemistry.

[45]  C. Richardson,et al.  The Linker Region between the Helicase and Primase Domains of the Bacteriophage T7 Gene 4 Protein Is Critical for Hexamer Formation* , 1999, The Journal of Biological Chemistry.

[46]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[47]  Eugene V. Koonin,et al.  Helicases: amino acid sequence comparisons and structure-function relationships , 1993 .

[48]  R. Kolodner,et al.  Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[49]  L. Romano,et al.  Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7. , 1989, The Journal of biological chemistry.

[50]  Edward H. Egelman,et al.  The RecA hexamer is a structural homologue of ring helicases , 1997, Nature Structural Biology.

[51]  J. Abrahams,et al.  The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis. , 1999, Biochemical Society transactions.

[52]  Irene T. Weber,et al.  The structure of the E. coli recA protein monomer and polymer , 1992, Nature.

[53]  Hu Pan,et al.  Characterization and crystallization of the helicase domain of bacteriophage T7 gene 4 protein , 1997, Nucleic Acids Res..

[54]  C. Richardson,et al.  The nucleotide binding site of the helicase/primase of bacteriophage T7. Interaction of mutant and wild-type proteins. , 1993, The Journal of biological chemistry.

[55]  Jones Ta,et al.  Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. , 1985, Methods in enzymology.

[56]  Z. Debyser,et al.  Coordination of leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 , 1994, Cell.

[57]  Smita S. Patel,et al.  Asymmetric Interactions of Hexameric Bacteriophage T7 DNA Helicase with the 5′- and 3′-Tails of the Forked DNA Substrate* , 1997, The Journal of Biological Chemistry.

[58]  C. Richardson.  Bacteriophage T7: Minimal requirements for the replication of a duplex DNA molecule , 1983, Cell.

[59]  E. Egelman,et al.  DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase , 1996, Nature Structural Biology.

[60]  L. Lally.  The CCP 4 Suite — Computer programs for protein crystallography , 1998 .

[61]  N. Ellis.  DNA helicases in inherited human disorders. , 1997, Current opinion in genetics & development.