The experiments described here demonstrate ways in which DNA length can be used as an experimental variable for the characterization of positively cooperative, sequence nonspecific DNA binding. Examples are drawn from recent studies of the interactions of O(6)-alkylguanine DNA alkyltransferase (AGT) with duplex DNAs (Melikishvili et al. (2008). Interactions of human O(6)-alkylguanine-DNA alkyltransferase (AGT) with short double-stranded DNAs. Biochemistry 47, 13754-13763). Oscillations in binding density and apparent binding site size (S(app)) are predicted by models in which a single cooperative assembly forms on each DNA molecule and in which enzyme molecules bind full-length binding sites, but not partial ones. These oscillations provide an accurate, DNA-length independent measure of the occluded binding site size (the length of DNA that one protein molecule occupies to the exclusion of others). In addition, length-dependent oscillations in association constant (K) and cooperativity (ω) reveal the degree to which substrate length can influence these parameters.
|Number of pages||17|
|Journal||Methods in Enzymology|
|State||Published - 2009|
Bibliographical noteFunding Information:
We gratefully acknowledge valuable discussions with Drs. Jack Correia, Michael Johnson, Jacob Lebowitz, James Cole, and Nichola Garbett. This work was supported by NIH grant GM-070662 to M. G. F.
ASJC Scopus subject areas
- Molecular Biology