Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.
The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.
Morrical
S W
SW
Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco 94143-0448.
Wong
M L
ML
Alberts
B M
BM
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D009695:Nucleic Acid Renaturation; D011995:Recombination, Genetic; D013604:T-Phages / Q000201:enzymology*; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C092863:UvsX protein, Enterobacteria phage T4; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases