Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states

Red beta is a single strand annealing protein from bacteriophage lambda that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Red beta forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Red beta in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Red beta forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Red beta in cells active for recombination and find it to range from 7 to 27 mu M. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.

Automated summary

This study describes the research methods and results of oligomer formation of Red beta protein in different DNA binding states, as well as the measurement of its concentration in cells. Various techniques were used to characterize the oligomers formed by Red beta, revealing the dynamic nature on ssDNA and the stable complex on annealed duplex. The study provides new insights into the functional forms of Red beta in vivo.