Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation.
DNA-protein interactions, mainly DNA-histone interactions, are thought to play an essential role in the packing mechanisms of chromatin as well as in transcriptional control. In this context it is important to develop new methods to study DNA-protein interactions and structural changes associated with them. Paraformaldehyde (PFA) has been shown to crosslink proteins, mainly histones, to DNA; and in this short study we have used a system with the DNA-binding dye 7-aminoactinomycin D (7-AAMD) as an indirect probe for PFA fixation. The aim was to investigate the dynamics of fixation on the binding of this dye to intact human lymphocytes. The results show a decrease in accessibility of 7-AAMD,initially affecting both the nonspecific binding of 7-AAMD and the high affinity binding sites, and thereafter mainly the high affinity binding sites. We conclude that fixation with PFA is a long-term reaction that requires a fixation time of several hours at a sufficient concentration to be completed. Our findings suggest that staining with a low concentration of 7-AAMD after extensive PFA fixation may be used to obtain information on DNA-protein interactions in intact cells.
Lindén
E
E
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
Skoglund
P
P
Rundquist
I
I
D001665:Binding Sites; D002467:Cell Nucleus; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005404:Fixatives / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008214:Lymphocytes; D011108:Polymers / Q000737:chemistry; D016707:Tissue Fixation / Q000379:methods*
D003432:Cross-Linking Reagents; D005404:Fixatives; D005456:Fluorescent Dyes; D011108:Polymers; D003609:Dactinomycin; D005557:Formaldehyde; C025942:7-aminoactinomycin D; D004247:DNA; C003043:paraform