The labeled medium was replaced with fresh medium, and the cells were grownto confluence.Pr ior to UV damage, cells were treated with rnM sodium butyrate for h.The cells were then cooled on ice, exposed to J m UV light, and harvested immediately.In all cases, nucleosome core DNA samples were obtained by proteinase K digestion and ethanol precipitation.The percent <a href="http://www.targetmol.com/compound/Arbidol-hydrochloride">sell
Arbidol hydrochloride</a> thymine present as cyclobutane thymine dimers was determined from the to talH radioactivity pre sent in the thymine dimer and thymine peaks.Nucleosomes were fractionated using two different published procedures.During the H labeling period, only repair patches are labeled since the low level of replicative synthesis normally detected in these con fluent cultures is abolished by thebutyrate trea tment was subjected to gel electrophoresis, under nondenaturing conditions, and the major components of thesample were mononucleosomes, with some di and trinucleosomes also present. Furthermore, the mon onucleosome population was comprised of three broad bands R DNA in these bands in a seconddimension gel demonstrated th at theslowest migrating band contained bp DNA contained of the starting mate rial, and the mononucleosomes present were of the total.Thehistone profiles obtained for the different nucleosome subpopulations indicated th at themon onucleosome species M contained a much larger fraction of hyperacetylated histone H. Therelative amount of repair synthesis present in theMI and M species was determined by excising the bp bands from the seconddimension DNA gel, slicing the bands, and assaying the slices for radioactivity.MI, M, and M designate the positions of three closely migrating mononucleosome species, and D designates the position of nucleosome dimer species.B, twodimensional electrophoresis of nucleosomes in the first dimension DNA in the second dimension.After silver staining, the gels were scanned on a laser densitometer.Profiles correspond to histones obtained from the fast. The peaks designated, I, and represent the different acetylated forms of histone H.Oligonucleosomes, and redigested with staphylococcal nu clease.L, with the and bp fragments denoted in the figure.C, gel profiles of histones from nucleosome cores of S, L, and P fractions.The gel profiles ob ta ined were an a lyzed as before and ind ica te thatfraction S conta ins thehighestlevel of hyperace ty la ted histones, whereas fractions L and P con ta in modera te and low levels of acetylated histones, respectively. The extent of repair syn thesis in the diffe rentnucleosome subpopu lations was de term ined by measuringtheHC ratio for each sample.Mono and dinucleosomes were, nuclei were digestedwi th staphylococcal nu ob ta ined from furtherstaphylococcal nuclease digestion of clease, and the ch romatin fragments released were fraction theoligonucleosome fractions, followed by separation on su crosegrad ients and gel electrophoresis.Af ter removal ofhistone H, the syn thes is inthehyperacetylated fraction S.However, when theaverage HC values in core DNA for several experiments are compared, there appears to be an increase in repair synthesis associated with nucleosome cores of the S fraction. Oligonucleosomes and nucleo some cores were obtained from further staphylococcal nuclease digestion of the oligonucleosome fractions, followed by separation on sucrose gradients and subsequent electrophoresis.We have also obtained S, L, and P fractions from cells not exposed to mM butyrate and either treated or not treated with mM hydroxyurea prior to UV damage.