Strong, positively cooperative binding can lead to the clustering of proteins on DNA. Here, we describe one approach to the analysis of such clusters. Our example is based on recent studies of the interactions of O6-alkylguanine DNA alkyltransferase (AGT) with high-molecular-weight DNAs (Adams et al., 2009; Tessmer, Melikishvili, & Fried, 2012). Cooperative cluster size distributions are predicted using the simplest homogeneous binding and cooperativity (HBC) model, together with data obtained by sedimentation equilibrium analysis. These predictions are tested using atomic force microscopy imaging; for AGT, measured cluster sizes are found to be significantly smaller than those predicted by the HBC model. A mechanism that may account for cluster size limitation is briefly discussed.
|Title of host publication||Methods in Enzymology|
|Number of pages||18|
|State||Published - 2015|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
These studies were supported by the Deutsche Forschungsgemeinschaft (DFG, Forschungszentrum FZ82 to I.T.) and National Institutes of Health (NIH, GM-070662 to M.G.F.).
© 2015 Elsevier Inc.
- Analytical ultracentrifugation
- Atomic force microscopy
- Cluster size analysis
- DNA binding
- Sedimentation equilibrium
ASJC Scopus subject areas
- Molecular Biology