Efficient protection against oxidative DNA damage in chromatin

Abstract

The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(II)-ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical-induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14-fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of "native" chromatin and "condensed" chromatin contained 100-fold and 300-fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage.

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

“…Instead, groups have been using ␥-rays (47) and x-rays (24,48) to generate hydroxyl radicals in vivo. The group utilizing x-rays noted that a 10-fold higher dose was required to obtain the same level of strand cleavage as obtained in vitro, in support of previous studies showing that chromatin greatly protects DNA from hydroxyl radical damage (49,50). We found that the Fe-EDTA system gave the same cleavage pattern for the FOS promoter nucleosome in HeLa cells as produced by x-rays in TIG-3 cells (24).…”

Protection of DNA against Direct Radiation Damage by Complex Formation with Positively Charged Polypeptides

“…The experiment with the DNA-PLT films sheds some light on the possible involvement of mechanism (2). Thermodynamically tyrosine will compete with guanine as a hole trap.…”

“…This DNA-protein complex has long been known to confer protection of DNA against radiation damage (1). Radiosensitivity of DNA, quantified as the yield of double-strand breaks (DSBs), was demonstrated to increase on going from intact cells to naked DNA, with the partially stripped chromatin being intermediate in this order (2,3). Stepwise removal of DNAbound proteins from the chromatin increases the amount of radiation-induced DSBs (4,5), with removal of core histones having the largest impact on the radiosensitivity of DNA (5).…”

Purification of protein–DNA complexes by native gel electrophoresis for electron microscopy study

“…Пе-рестройки хроматина, сборка и разборка нуклеосом сопровождают все процессы, происходящие с учас-тием генетического материала в клетке. Нарушения процессов ДНК-гистоновых и гистон-гистоновых взаимодействий и необратимая потеря нуклеосом в результате действия различных факторов способ-ны запускать патологические процессы и снижать жизнеспособность клеток, приводя, в частности, к блокировке процессов транскрипции [7][8][9].…”

“…№ 1. C. [3][4][5][6][7][8] нена в связи с большими размерами и структурной лабильностью. Поэтому основным методом полу-чения структурной информации в данном случае служит просвечивающая электронная микроскопия (ПЭМ) макромолекул [10].…”