A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing.
The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lisa F
LF
Rezende
Smaranda
S
Willcox
Jack D
JD
Griffith
Charles C
CC
Richardson
D017123:Bacteriophage T7 / Q000737:chemistry*; D003460:Crystallization; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000821:virology; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase