The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein-DNA complexes.

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.

[1]  K. V. van Holde,et al.  Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1. , 1989, Biochemistry.

[2]  K. V. van Holde,et al.  Binding of the RNA polymerase I transcription complex to its promoter can modify positioning of downstream nucleosomes assembled in vitro. , 1993, The Journal of biological chemistry.

[3]  T. Richmond,et al.  DNA-dependent divalent cation binding in the nucleosome core particle , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[4]  G. Hager,et al.  Formation of higher-order secondary and tertiary chromatin structures by genomic mouse mammary tumor virus promoters. , 2003, Genes & development.

[5]  F. Thoma,et al.  Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: A model system for study of higher order structure , 1985, Cell.

[6]  Ilya Ioshikhes,et al.  Structural dynamics of nucleosome core particle: Comparison with nucleosomes containing histone variants , 2004, Proteins.

[7]  P. Serwer,et al.  Quantitative agarose gel electrophoresis of chromatin: nucleosome-dependent changes in charge, sharp, and deformability at low ionic strength. , 1994, Biochemistry.

[8]  Dominique Durand,et al.  Salt-induced conformation and interaction changes of nucleosome core particles. , 2002, Biophysical journal.

[9]  D. Lohr,et al.  Assembly and structural properties of subsaturated chromatin arrays. , 1993, The Journal of biological chemistry.

[10]  P. Serwer,et al.  The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling , 1989, Biopolymers.

[11]  P. Georgel,et al.  Characterization of chromatin samples in the presence of Drosophila embryo extract by quantitative agarose gel electrophoresis. , 2006, Journal of biochemical and biophysical methods.

[12]  H. Eisenberg,et al.  Nucleosome core particle stability and conformational change. Effect of temperature, particle and NaCl concentrations, and crosslinking of histone H3 sulfhydryl groups. , 1984, Journal of molecular biology.

[13]  Karolin Luger,et al.  Nucleosome core particles containing a poly(dA.dT) sequence element exhibit a locally distorted DNA structure. , 2006, Journal of molecular biology.

[14]  P. Georgel,et al.  Biophysical analysis of specific genomic loci assembled as chromatin in vivo. , 2004, Methods in enzymology.

[15]  A. Wolffe,et al.  Disruption of Reconstituted Nucleosomes , 1995, The Journal of Biological Chemistry.

[16]  J. Hansen,et al.  Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N-termini in chromatin fiber compaction. , 1997, Biochemistry.

[17]  P. Serwer.  Sieving by agarose gels and its use during pulsed-field electrophoresis. , 1990, Biotechnology & genetic engineering reviews.

[18]  P. Serwer,et al.  Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: application to chromatin folding. , 1994, Biochemistry.

[19]  K. V. van Holde,et al.  Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Georgel,et al.  Quantitative characterization of specific genomic promoters using agarose gel electrophoresis. , 2003, Biopolymers.

[21]  Andrew J. Spakowitz,et al.  Effect of force on mononucleosomal dynamics , 2006, Proceedings of the National Academy of Sciences.