title: Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin.
abstract: Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. barak_akabayov@hms.harvard.edu
authors.forename: Barak
authors.initials: B
authors.lastname: Akabayov
authors.forename: Sabine R
authors.initials: SR
authors.lastname: Akabayov
authors.forename: Seung-Joo
authors.initials: SJ
authors.lastname: Lee
authors.forename: Stanley
authors.initials: S
authors.lastname: Tabor
authors.forename: Arkadiusz W
authors.initials: AW
authors.lastname: Kulczyk
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D053838:Scattering, Small Angle; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D013879:Thioredoxins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase