Characterization of Homogeneous, Cooperative Protein-DNA Clusters by Sedimentation Equilibrium Analytical Ultracentrifugation and Atomic Force Microscopy

Ingrid Tessmer, Michael G. Fried

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
Pages331-348
Number of pages18
DOIs
StatePublished - 2015

Publication series

NameMethods in Enzymology
Volume562
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Publisher Copyright:
© 2015 Elsevier Inc.

Keywords

  • Analytical ultracentrifugation
  • Atomic force microscopy
  • Cluster size analysis
  • Cooperativity
  • DNA binding
  • Sedimentation equilibrium

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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