Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen

Abstract

Intracellular simian virus 40 (SV40) chromatin was photoreacted with a 3H-labeled psoralen derivative, hydroxymethyltrimethylpsoralen (HMT), at 48 h postinfection. Psoralen compounds have been shown to readily penetrate intact cells and, in the presence of long-wavelength UV light, form covalent adducts to DNA, preferentially at regions unprotected by nucleosomes. The average distribution pattern of [3H]HMT on the SV40 genome was determined by specific activity measurements of the DNA fragments generated by HindIII plus HpaII or by AtuI restriction enzyme digestion. At levels of 1 to 10 [3H]HMT photoadducts per SV40 molecule, the radiolabel was found to be distributed nonrandomly. Comparison of the labeling pattern in vivo with that of purified SV40 DNA labeled in vitro revealed one major difference. A region of approximately 400 base pairs, located between 0.65 and 0.73 on the physical map, was preferentially labeled under in vivo conditions. This finding strongly suggests that the highly accessible region near the origin of replication, previously observed on isolated SV40 "minichromosomes," exists on SV40 chromatin in vivo during a lytic infection.