A benchmark for chromatin binding measurements in live cells
Top Cited Papers
Open Access
- 25 July 2012
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 40 (15), e119
- https://doi.org/10.1093/nar/gks701
Abstract
Live-cell measurement of protein binding to chromatin allows probing cellular biochemistry in physiological conditions, which are difficult to mimic in vitro. However, different studies have yielded widely discrepant predictions, and so it remains uncertain how to make the measurements accurately. To establish a benchmark we measured binding of the transcription factor p53 to chromatin by three approaches: fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking (SMT). Using new procedures to analyze the SMT data and to guide the FRAP and FCS analysis, we show how all three approaches yield similar estimates for both the fraction of p53 molecules bound to chromatin (only about 20%) and the residence time of these bound molecules (∼1.8 s). We also apply these procedures to mutants in p53 chromatin binding. Our results support the model that p53 locates specific sites by first binding at sequence-independent sites.Keywords
This publication has 52 references indexed in Scilit:
- p53 Requires an Intact C-Terminal Domain for DNA Binding and TransactivationJournal of Molecular Biology, 2012
- Activated STAT1 Transcription Factors Conduct Distinct Saltatory Movements in the Cell NucleusBiophysical Journal, 2011
- A Quantitative Approach to Analyze Binding Diffusion Kinetics by Confocal FRAPBiophysical Journal, 2010
- Cross-Validating FRAP and FCS to Quantify the Impact of Photobleaching on In Vivo Binding EstimatesBiophysical Journal, 2010
- Balbiani Ring mRNPs Diffuse through and Bind to Clusters of Large Intranuclear Molecular StructuresBiophysical Journal, 2010
- FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know?Current Opinion in Cell Biology, 2010
- Statistical Analysis of Lateral Diffusion and Multistate Kinetics in Single-Molecule ImagingBiophysical Journal, 2009
- Direct Measurement of Association and Dissociation Rates of DNA Binding in Live Cells by Fluorescence Correlation SpectroscopyBiophysical Journal, 2009
- In Vivo Labeling Method Using a Genetic Construct for Nanoscale Resolution MicroscopyBiophysical Journal, 2009
- Methods of Digital Video Microscopy for Colloidal StudiesJournal of Colloid and Interface Science, 1996