Topoisomerase 1

Chromatin assembly grade wheat germ extract (WEPRO7240Ch, CellFree Sciences) was used to co-express all core histone combinations using approximately 2 μg each histone mRNA^{31 (link)}. As a chromatin assembly control, 2 μg each of canonical H3 or variant H3 and H4-coding mRNA was used for the reaction. Briefly, individually transcribed and purified mRNAs were used to make 5 μL premix mRNA solutions for each chromatin combination and were mixed with 5 μL wheat germ extract, 0.4 μL of 1 mg·mL⁻¹ creatine kinase, 0.5 μL of 0.5 μg·μL⁻¹ pBSK plasmid, 0.1 μL of 20 U·μL⁻¹ Topoisomerase I (Takara, Shiga, Japan), and adjusted with nuclease-free water to 10.7 μL. Topoisomerase I was added in order to release torsional strain during chromatin assembly onto circular closed plasmid. The reaction mixture was carefully layered under 103 μL of 1xSUB-AMIX (CellFree Sciences) to create a bilayer in a sterile microplate well. The chromatin assembly reaction was performed for 4 h at 26 °C.

Glycyl-L-Glycine (HGlyGly) was purchased from Fluka (Switzerland); Glycyl-L-Alanine (HGlyAla) from Roanal (Hungary); Glycyl-L-Aspartic acid (H₂GlyAsp); calf thymus DNA (CT DNA), Hoechst 33258, plasmid DNA (pBR322), tris(hydroxymethyl)aminomethane (Tris), propidium iodide (PI), phosphate buffered saline (PBS), agarose and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemie (Germany); ethidium bromide (EB) from Boehringer Mannheim (Germany); topoisomerase I from Takara (Japan); cisplatin from Acros Organics (USA); and silver(I) nitrate from Lachema (Czech Republic). topoisomerase I was purchased from Inspiralis. Other chemicals were analytically pure and used without purification.

General reagents used in cDNA synthesis (Nippon Genetics), cloning (Toyobo), in vitro transcription (Promega), protein expression, including wheat germ extract (WEPRO7240H Lot: 15AQ02, CellFree Sciences) and SUB-AMIX translation buffer (CellFree Sciences), and creatine kinase (Roche Diagnostics, GmbH) were purchased. Reagents used in chromatin assembly reaction, including pBluescript II SK plasmid DNA (Addgene), SUB-AMIX solution (CellFree Sciences), creatine kinase (Roche Diagnostics, GmbH), and Topoisomerase I (Takara Bio Inc.) were also purchased. For gel analyses,100 bp DNA molecular weight marker (Toyobo) and protein size marker (Bio-Rad Laboratories) were purchased.