A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor.
Bacteriophage T7 DNA polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase/primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer/template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA polymerase with other proteins of the replisome.
Hamdan
Samir M
SM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Marintcheva
Boriana
B
Cook
Timothy
T
Lee
Seung-Joo
SJ
Tabor
Stanley
S
Richardson
Charles C
CC
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005814:Genes, Viral; D008954:Models, Biological; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D017384:Sequence Deletion; D013441:Sulfinic Acids; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D013441:Sulfinic Acids; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C100040:thiosulfinic acids; D013879:Thioredoxins; D019915:DNA Primase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase