Native chromatin and damage induced by nuclease

Area of Science:

• Molecular Biology
• Biophysics
• Chromatin Structure

Background:

• Understanding chromatin structure is crucial for comprehending DNA packaging and gene regulation.
• Previous methods for chromatin preparation, including limited nuclease digestion, may introduce structural artifacts.
• The in situ structure of chromatin within native nuclei serves as a benchmark for assessing preparation techniques.

Purpose of the Study:

• To evaluate the structural integrity of chromatin prepared using different methods.
• To compare the characteristics of prepared chromatin with native nuclear chromatin.
• To identify reliable methods for preserving the native chromatin-DNA structure.

Main Methods:

• Differential scanning calorimetry (DSC) to assess thermal stability.
• Gel electrophoresis to determine DNA fragment size and integrity.
• Polarized light scattering (PLS) to analyze quaternary structure and nucleofilament length.
• Studies were conducted at varying ionic strengths (low and high) and after shearing.

Main Results:

• Chromatin prepared by limited nuclease digestion exhibited artefactual structures, differing significantly from native nuclei.
• Chromatin prepared by lysis of native nuclei, followed by sedimentation and suspension, preserved the in situ structure.
• Native nucleofilaments were observed to be longer than 200 nucleosomes and possessed a stable quaternary structure, even at 0.01 M ionic strength.

Conclusions:

• Limited nuclease digestion is not a suitable method for preparing chromatin that accurately reflects its native state.
• Nuclear lysis and subsequent manipulation are superior methods for preserving the native chromatin-DNA structure and quaternary organization.
• Native chromatin exhibits remarkable structural stability, maintaining its integrity under various conditions.