Chromatin Studies by DNA–Protein Cross-Linking

Nucleosome Structural Transition during Chromatin Unfolding Is Caused by Conformational Changes in Nucleosomal DNA

“…Occurring during Chromatin Unfolding-To study the histone-DNA interactions in isolated core particles under different ionic conditions, we used the method of chemically induced zero-length protein-DNA cross-linking (31,33). Cross-linking causes a single-stranded nick of the nucleosomal DNA at the site of cross-linking such that only the 5Ј-terminal DNA fragment becomes attached to a histone molecule (33).…”

“…Cross-linking causes a single-stranded nick of the nucleosomal DNA at the site of cross-linking such that only the 5Ј-terminal DNA fragment becomes attached to a histone molecule (33). Therefore, the length of the DNA fragment cross-linked to a particular histone is the precise distance of a protein cross-linking site from the 5Ј-end of the nucleosomal DNA and can be assessed by the two-dimensional gel electrophoresis.…”

“…Therefore, the length of the DNA fragment cross-linked to a particular histone is the precise distance of a protein cross-linking site from the 5Ј-end of the nucleosomal DNA and can be assessed by the two-dimensional gel electrophoresis. To map the histone-DNA contacts in nucleosome core particles under different ionic conditions, we have used a protein version of the two-dimensional gel electrophoresis system (31,33). In the denaturing first dimension SDS gel, the mobility of 125 I-labeled histone-DNA cross-linked complexes depends on the molecular weight of the histones and the size of the cross-linked DNA fragment.…”

“…In the denaturing first dimension SDS gel, the mobility of 125 I-labeled histone-DNA cross-linked complexes depends on the molecular weight of the histones and the size of the cross-linked DNA fragment. After separation of cross-linked complexes in the first dimension, the DNA is hydrolyzed directly in the gel according to a modified Burton's procedure (31,33,34), and released 125 Ilabeled histones are separated in the second dimension SDS gel (35) according to their size. In the first dimension, the mobility of histones is decreased proportionally to the size of DNA fragments cross-linked to a particular histone (33).…”

“…After separation of cross-linked complexes in the first dimension, the DNA is hydrolyzed directly in the gel according to a modified Burton's procedure (31,33,34), and released 125 Ilabeled histones are separated in the second dimension SDS gel (35) according to their size. In the first dimension, the mobility of histones is decreased proportionally to the size of DNA fragments cross-linked to a particular histone (33). As a result, in the gel of the second dimension, the 125 I-labeled histones are arranged as spots on different horizontal lines ( Figs.…”