Question

SAR/MAR sequences were postulated to form the bases of these radial loops.  SAR/MAR sequences were defined experimentally by one approach for SARs and by a different experimental approach for MARs. This question is about our experimental foundation for thinking about models for chromosome folding and nuclear organization as discussed in class which lead to the idea of a radial loop model of chromosome origination. SAR/MAR sequences were postulated to form the bases of these radial loops.  SAR/MAR sequences were defined experimentally by one approach for SARs and by a different experimental approach for MARs. SAR/MAR sequences later turned out to be enriched in DNA sequences that show high binding affinity for topoisomerase 2.  Attention then turned to identifying proteins that bind to SAR/MAR sequences in mitotic chromosomes.  One protein in fact was found to be topoisomerase 2.

The radial loop model was criticized because harsh conditions were used in the experimental approaches taken to identify both SAR sequences and proteins binding to SARs.

What experimental approach was taken to support the idea that both topoisomerase 2 and the other SAR binding protein bind the bases of the radial loops under more physiological conditions?

Answer 1

The experimental approach that can support the interaction between the topoisomerase 2 along with SAR binding protien and the DNA bases of the radial loop is Chromatin immunoprecitation assay. This assay fixes the invivo interaction by means of using a fixative like formaldehyde which can maintain the physiological conditions as such. It captures a snapshot of specific protein–DNA interactions as they occur in living cells.

Below are the steps involved in CHIP

DNA-binding proteins areUse PCR to amplify specific DNA sequences to see if they were precipitated with the antibody. crosslinked to DNA with formaldehyde in vivo.

Isolate the chromatin by shearing the DNA into tiny fragments along with attached proteins.

Link DNA-binding protein-specific antibodies to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins