A 1.0-kb DNA fragment from the end of the mouse gene described in the previous problem is examined by DNA footprint protection analysis (see Research Technique 8.1). Two samples are end-labeled with ³²P and one of the two is mixed with TFIIB, TFIID, and RNA polymerase II. The DNA exposed to these proteins is run in the right-hand lane of the gel shown below and the control DNA is run in the left-hand. Both DNA samples are treated with DNase I before running the samples on the electrophoresis gel. Explain the role of DNase I.

Key Concepts

DNase I Function

DNase I is an enzyme that cleaves the phosphodiester bonds between nucleotides in DNA, leading to the degradation of DNA into smaller fragments. In the context of DNA footprinting, DNase I is used to selectively digest unprotected regions of DNA, allowing researchers to identify which parts of the DNA are bound by proteins. This helps in understanding protein-DNA interactions and the regulatory mechanisms of gene expression.

DNA Footprinting

DNA footprinting is a technique used to study the binding of proteins to specific DNA sequences. By treating DNA with DNase I in the presence of bound proteins, the enzyme will only cleave the unprotected regions, creating a 'footprint' that indicates where the proteins are attached. The resulting fragments are then analyzed through gel electrophoresis, revealing the locations of protein binding sites on the DNA.

Electrophoresis

Electrophoresis is a laboratory technique used to separate charged molecules, such as DNA fragments, based on their size and charge. In this process, DNA samples are placed in a gel matrix and subjected to an electric field, causing the fragments to migrate through the gel. Smaller fragments move faster and travel further than larger ones, allowing for the visualization and analysis of the DNA fragments generated by DNase I digestion in the footprinting experiment.