P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.
Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S. Chen and M.T.Hsu, J. Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments.
Chu
Y
Y
Department of Microbiology, Mt Sinai Medical Center, New York, NY 10029.
Huang
T S
TS
Hsu
M T
MT
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D013539:Simian virus 40 / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
D002843:Chromatin; D004279:DNA, Viral; D003851:Deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases