Polynucleosomal synthesis of poly(ADP-ribose) causes chromatin unfolding as determined by micrococcal nuclease digestion.
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 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), 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 Mg(2+) 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 microM betaNAD(+). 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.
Perez-Lamigueiro
Maria A
MA
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
Alvarez-Gonzalez
Rafael
R
D000818:Animals; D002417:Cattle; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis*; D011489:Protein Denaturation
D002843:Chromatin; D009707:Nucleosomes; D011064:Poly Adenosine Diphosphate Ribose; D008836:Micrococcal Nuclease