This research proposal seeks (1) to quantitate, using UV irradiation, the changes in histone/DNA crosslinking caused by perturbants which are known to induce varying degrees of conformational changes in chromatin. These studies will be used to evaluate the nature of "altered" chromatin in which the histones have been modified in vivo by acetylation or phosphorylation; (2) to examine the size and number of histone to DNA binding domains by fingerprinting the histone/DNA crosslinked peptides; and (3) to purify and characterize the peptides to gain insight into the molecular nature of the histone to DNA interacting domains. The specific aims to achieve the above objectives can be listed as follows: (I) To determine crosslinking rate constants, in order to evaluate the extent of UV-induced histone/DNA crosslinking as a function of chromatin perturbations by (a) nuclease treatment, (b) salt and urea treatment, (c) stepwise reconstitution of nucleosomes, (d) in vivo acetylation of the core histones and phosphorylation of the linker histones; (II) To purify and fingerprint the UV-induced peptide/DNA crosslinks; to reconstitute, and identify the interrelationships of tryptic, thermolytic and chymotryptic fingerprints; (III) To obtain crosslinking peptide maps of perturbed chromatin in an effort to further evaluate the conformation contributions of the modifiers: (IV) To characterize the crosslinked peptides by (a) gas chromatography mass spectrometry (GCMS) of small tryptic peptides, (b) a new mass spectrometry "fingerprinting" sequencing technique to determine amino acid sequence of the peptides, and (c) conventional manual and automatic Edman degradation techniques to determine the amino acid sequence of the peptides.
NIH ExPorter
NIH ExPorter