Polynucleosomal Synthesis of Poly(ADP-ribose) Causes Chromatin Unfolding as Determined by Micrococcal Nuclease Digestion

• Published in: Annals of the New York Academy of Sciences 2004-12, Vol.1030 (1), p.593-598
• Main Authors: PEREZ-LAMIGUEIRO, MARIA A., ALVAREZ-GONZALEZ, RAFAEL
• Format: Article
• Language: English
• Subjects: agarose gel electrophoresis; Animals; benzamide; Cattle; Chromatin - chemistry; Chromatin - metabolism; chromatin structure; Hydrolysis; Kinetics; micrococcal nuclease; Micrococcal Nuclease - metabolism; Nucleosomes - metabolism; Poly Adenosine Diphosphate Ribose - biosynthesis; Protein Denaturation; protein poly(ADP-ribosyl)ation
• ISSN: 0077-8923; 1749-6632
• DOI: 10.1196/annals.1329.069

: We have evaluated the influence of protein poly(ADP‐ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein‐free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 μM βNAD+, the poly(ADP‐ribosyl)ation substrate, before MNase digestion. For incubations in the presence of βNAD+, the synthesis of polynucleosomal poly(ADP‐ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg2+ ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP‐ribosyl)ation when the chromatin extract was preincubated with 200 μM βNAD+. Thus, our results are consistent with the conclusion that the covalent poly(ADP‐ribosyl)ation of polynucleosomal proteins favors a more “relaxed” or “open” structure, which renders chromatin more susceptible to MNase digestion.