Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents.
To investigate the in vivo effects of macromolecular crowding we examined the effect of inert macromolecules such as polyvinyl alcohol and polyethylene glycol on the in vitro activity of recA protein. The addition of either of these volume-occupying agents enables recA protein to promote homologous pairing and exchange of DNA strands at an otherwise nonpermissive magnesium ion concentration. In the presence of these macromolecules, both the rate of recA protein association with single-stranded DNA (ssDNA) and the steady-state affinity of recA protein for ssDNA are increased. Consequently, the ability of recA protein to compete with ssDNA-binding protein (SSB protein) is enhanced, and the inhibitory effects of SSB protein on the formation of recA protein-ssDNA presynaptic complexes are eliminated. Because the ability of recA protein to bind to ssDNA-containing secondary structures is also enhanced in volume-occupied solution, joint molecule formation is not greatly reduced when SSB protein is omitted from the reaction. Thus, increased recA protein interactions with ssDNA contribute to enhanced presynaptic complex formation. In addition, polyvinyl alcohol and polyethylene glycol must also affect another property of recA protein, i.e. self-association, which is required for synapsis and DNA strand exchange. Our examination of DNA strand exchange in the presence of volume-occupying agents helps to reconcile the requirement for elevated magnesium ion concentrations in recA protein-promoted recombination reactions in vitro, with a presumably low magnesium ion concentration in vivo.
Lavery
P E
PE
Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Kowalczykowski
S C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011142:Polyvinyl Alcohol / Q000494:pharmacology; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D011142:Polyvinyl Alcohol; D004247:DNA; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D008274:Magnesium