Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency

Abstract

Nucleosome core particles (NCPs) are basic units of chromatin organization; they represent the most convenient model system for the study of key DNA-dependent processes. Therefore, a robust method of nucleosome assembly is important for research. To prepare NCPs, purified histones and DNAs with sequences providing strong positioning of DNA relative to histone octamer are commonly used, and a method to control the efficacy of NCP reconstruction is required. Aim. To optimize the procedure for NCP reconstitution from purified histone octamers and different types of synthetic model DNAs and develop a new approach for express analysis of the efficacy of NCP reconstitution. Methods. Dialysis, PAAG, fluorescence measurement using the Eva-Green dye. Results. We first developed a convenient procedure for NCP assembly in a low-salt buffer with a variable DNA-histone ratio at the first stage. Once the optimal ratio was determined, NCPs could be assembled by a slow gradient dialysis. The efficacy of NCP assembly can be estimated directly in the solution using the Eva-Green dye. Conclusions. The efficacy of dye intercalation into DNA duplex was sharply reduced in the nucleosomal context. The main benefits of the proposed approach are the rapid analysis directly in the solution and possibility to use DNAs without any special tags.

Regulation of Poly(ADP-Ribose) Polymerase 1 Activity by Y-Box-Binding Protein 1

“…A histone H1 and bovine serum albumin (BSA) were purchased from Sigma (Sigma-Aldrich, Saint–Louse, MO, USA). Core histones (H2A, H2B, H3, and H4) were purified from chicken erythrocytes as described in [ 39 ]. The fragment of YB-1 containing an alanine/proline-rich N-terminal domain and the cold shock domain (AP-CSD) was а kind gift from Drs.…”

“…The star denotes the position of the one-nucleotide gap formed after uracil-DNA glycosylase (UDG) and EndoIII treatment. Mononucleosomes were reconstituted by the salt-dialysis method, as described earlier [39]. riefly, an appropriate histone/DNA ratio was found experimentally by the quick-time constitution approach.…”

“…he PCR product (147 bp DNA) was ethanol precipitated and dissolved in double-distilled H2O. Mononucleosomes were reconstituted by the salt-dialysis method, as described earlier [39]. riefly, an appropriate histone/DNA ratio was found experimentally by the quick-time constitution approach.…”

“…riefly, an appropriate histone/DNA ratio was found experimentally by the quick-time constitution approach. The 147 bp DNA at a final concentration of 0.1 µM was mixed with histones f different concentrations in low-salt buffer (10 mM NaCl, 0.2 mM EDTA, 5 mM β-mercaptoethanol, .1% of NP-40, and 10 mM Tris-HCl pH 7.5), incubated for 15 min at 37 °C, and analysed by 4% olyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions (Supplementary Mononucleosomes were reconstituted by the salt-dialysis method, as described earlier [39]. Briefly, an appropriate histone/DNA ratio was found experimentally by the quick-time reconstitution approach.…”

The contribution of PARP1, PARP2 and poly(ADP-ribosyl)ation to base excision repair in the nucleosomal context

“…Oligodeoxyribonucleotides were 5′[ 32 P]phosphorylated by T4 polynucleotide kinase as described before (92). The amplification of 5′-FAM- or 32 P-labelled DNA and subsequent NCP reconstitution were performed according to (98). The purity of the sample was estimated by the homogeneity controlled by electrophoretic mobility under on a 10% polyacrylamide gel under non-denaturing conditions.…”