Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C-terminal phenylalanine.
Gene 2.5 of bacteriophage T7 encodes a ssDNA binding protein (gp2.5) essential for DNA replication. The C-terminal phenylalanine of gp2.5 is critical for function and mutations in that position are dominant lethal. In order to identify gp2.5 interactions we designed a screen for suppressors of gp2.5 lacking the C-terminal phenylalanine. Screening for suppressors of dominant lethal mutations of essential genes is challenging as the phenotype prevents propagation. We select for phage encoding a dominant lethal version of gene 2.5, whose viability is recovered via second-site suppressor mutation(s). Functional gp2.5 is expressed in trans for propagation of the unviable phage and allows suppression to occur via natural selection. The isolated intragenic suppressors support the critical role of the C-terminal phenylalanine. Extragenic suppressor mutations occur in several genes encoding enzymes of DNA metabolism. We have focused on the suppressor mutations in gene 5 encoding the T7 DNA polymerase (gp5) as the gp5/gp2.5 interaction is well documented. The suppressor mutations in gene 5 are necessary and sufficient to suppress the lethal phenotype of gp2.5 lacking the C-terminal phenylalanine. The affected residues map in proximity to aromatic residues and to residues in contact with DNA in the crystal structure of T7 DNA polymerase-thioredoxin.
Marintcheva
Boriana
B
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Qimron
Udi
U
Yu
Yao
Y
Tabor
Stanley
S
Richardson
Charles C
CC
Richardson
Charles
C
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004926:Escherichia coli / Q000821:virology; D010649:Phenylalanine / Q000235:genetics*; D013489:Suppression, Genetic; D014764:Viral Proteins / Q000235:genetics*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; D010649:Phenylalanine; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase