Measurement of localized DNA supercoiling and topological domain size in eukaryotic cells

Publisher Summary This chapter describes the process of measuring localized DNA supercoiling and topological domain size in eukaryotic cells. Psoralen has distinct advantages as an in vivo probe to measure accurately the level of unrestrained supercoiling. The main advantages of psoralen are (1) it is readily permeable to eukaryotic cells, (2) it has little effect on cells in the absence of long-wave (360 nm) ultraviolet (UV) light, (3) it will not displace nucleosomes, and (4) cells can survive high numbers of psoralen cross-links. Psoralen can also be used to estimate a localized topological domain size within living cells. The existence of unrestrained negative supercoiling is dependent on the partitioning of DNA into individual topological domains that have traditionally been envisioned as loops. Loops may be organized from folding a chromosome, as proposed for Escherichia coli , or from the periodic attachment of DNA to a nuclear scaffold, as suggested in eukaryotes, or attachment to the bacterial membrane in bacterial cells. This technique can determine an in vivo domain size on chromosomal DNA with its nucleosome organization and higher order organizational structure intact. For measurements of supercoiling in vivo , the rate of psoralen photo binding is dependent on both supercoiling and protein organization.