T4 dna ligase

The 3.4 kb DNA fragment of srp, a suppressor of recA polA lethality^{6 (link)}, was cloned into the BamHI site on pHSG576^{30 (link)}. The resultant plasmids were termed pSRO1. pSRO1, pSROΔhslO, and pSrpC were described previously^{6 (link)}. To construct pSROΔyrfG, pAQ10917 was digested with NsiI and BsmBI and self-ligated. In self-ligations, 1 μg digested DNA was blunted using 1 unit of T4 DNA polymerase (New England Bio Labs, Ipswich, MA, USA) supplemented with 1 mM dNTPS in NEBbuffer 1.1 at 12 °C for 20 min in 20 μL reaction, followed with heat inactivation at 75 °C for 30 min. Ethanol-precipitated blunted DNA was then ligated with 250 units of T4 DNA ligase (Nippon gene, Tokyo, Japan) in manufacturer-supplemented reaction buffer at 16 °C for 12 h. Then, a BamHI DNA fragment with a yrfG deletion was cloned into pHSG576. Subsequently, an spc cassette was cloned into the EcoRI site. The resultant plasmid was called pSROΔyrfG. To construct pSROΔhslRO, pAQ10917 was digested with BsmBI and BstEII and self-ligated. Subsequently, a BamHI DNA fragment containing a hslR to hslO deletion was cloned into pHSG576. Then, an spc cassette was cloned into the EcoRI site. The resultant plasmid was called pSROΔhslRO.

Three mono-nucleosomes with different sticky ends, 601a, 601b, and 601c, were reconstituted by salt dialysis, and were purified by 6% polyacrylamide gel electrophoresis, using a Prep Cell apparatus (Bio-Rad). The tri-nucleosome array was prepared by the ligation of these 601a, 601b, and 601c mono-nucleosomes. The di-nucleosome array was prepared by the ligation of the 601a mono-nucleosome, the DNA fragment containing the 601b sequence, and the 601c mono-nucleosome. The ligation reaction was conducted with T4 DNA ligase (Nippon Gene), and the resulting nucleosome arrays were further purified by 4% polyacrylamide gel electrophoresis, using a Prep Cell apparatus.

The NCPs were prepared by the salt dialysis method with the palindromic 146 base-pair α-satellite DNA fragment, as described previously^{1 (link),65 (link)}. The DNA fragment containing one half of the α-satellite DNA fragment in pGEM-T Easy vector was amplified in the E. coli strain DH5α, and was excised from the plasmid DNA by EcoR V (Takara). The DNA fragment was then dephosphorylated by alkaline phosphatase (Takara), and was further cleaved by EcoR I. The DNA fragment was purified by DEAE-5PW anion-exchange column chromatography (TOSOH). The DNA fragment was self-ligated by T4 DNA ligase (NIPPON GENE), and the resulting DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH).
The reconstituted NCPs were purified by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad). For the H2A/H2A.B heterotypic NCP preparation, the 146 base-pair DNA, the H2A-H2B dimer, the H2A.B-H2B dimer, and the H3.1-H4 tetramer were mixed in a 1:0.85:2.55:1.7 molar ratio. The NCPs were reconstituted by the salt dialysis method. As a result, H2A/H2A, H2A/H2A.B, and H2A.B/H2A.B NCPs were reconstituted. The resulting three types of NCPs were separated by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad), and the heterotypic NCP was selectively purified.