Bacteriophage T4 strand transfer protein UvsX tolerates symmetric and asymmetric heterologies in short double-stranded oligonucleotides.
The UvsX protein of bacteriophage T4 catalyzes strand transfer from double-stranded DNA to homologous single-stranded DNA to generate both paranemic and plectonemic joints. We demonstrate here that UvsX mediates strand transfer efficiently from synthetic double-stranded donor oligonucleotides of 30 to 117 bp in length to circular single-stranded recipient M13mp19 DNA. Recovery of a diagnostic BamHI-restriction site, activated in the recipient after strand transfer, demonstrates that recipient and donated strands are perfectly base-paired after the exchange reaction has taken place. The transfer reaction progresses with greatest efficiency using donor DNA with a 3' overhang. Use of donor DNA having recessed 3' ends or blunt ends reduces the transfer efficiency by half. Single-stranded heterologies, centrally located in either strand of the donor DNA and forming either heteroduplex loops or a bulge in the donor are transferred with 80 to 100% efficiency. Also, a centrally located C/C-mismatch in the donor does not affect the transfer efficiency. Double-stranded heterologies are tolerated by the UvsX-catalyzed reaction but have different effects on the transfer efficiencies, depending on length and location in the molecule. A heterology of 24 bp located at the proximal end (start of transfer), the distal end (termination of transfer) and at each end of the donor molecule results in transfer efficiencies of 100%, 50% and 50 to 60%, respectively. Strand transfer efficiency is markedly reduced to about 15% if the 24 bp heterology is at a central location. However, insertion of a 4 bp heterology at this position yields a transfer efficiency of about 30%. Also, large double-stranded heterologies of 187 bp at the proximal end or 590 bp at the distal end of control-donor DNAs derived from plasmid digests did not impair the transfer activity of UvsX. This result differs from published results obtained with strand transfer reactions with large distally located heterologies catalyzed by RecA of Escherichia coli in vitro.
Birkenkamp
K
K
Institut für Genetik der Universität zu Köln, Germany.
Kemper
B
B
D017122:Bacteriophage T4; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011938:Rec A Recombinases / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C092863:UvsX protein, Enterobacteria phage T4; D014764:Viral Proteins; D004247:DNA; D011938:Rec A Recombinases