C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.
Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.
Ghosh
Sharmistha
S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Marintcheva
Boriana
B
Takahashi
Masateru
M
Richardson
Charles C
CC
D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D009154:Mutation; D010649:Phenylalanine; D011485:Protein Binding; D012097:Repressor Proteins
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D010649:Phenylalanine; D004259:DNA-Directed DNA Polymerase