Stoichiometry of the binding of chromosomal protein MCl from the archaebacterium, Methanosarcina spp. CHTI55, to DNA

Abstract

We have investigated the binding stoichiometry of the chramosomal MCI protein on DNA using the gel reta~atio~ technique. Analysis of the distribution of the complex containing 0, I, 2, 3...... bound proteins shows that the protein MCI interacts with the DNA as a monomer. Binding experiments with short DNA fragments of various lengths shows that the site size is 11 bp in length. These results are compared to those obtained with other chromosomal proteins including HU protein. In vivo, the protein-to-DNA ratio is estimated to be I protein molecule for every 170 bp DNA. In this paper, gel retardation experiments enabled us to determine the DNA binding stoichiometry of MCI protein. The MC1 protein interacts with DNA in a monomeric state on a binding site which is of about 11 bp long. MATERIALS AND METHODS DNA frugmentsThe different DNA fragments (236,55,46,25 and 21 bp long) were obtained by electrophoresis on a 6% preparative polyacrylamide gel after restriction digestion of the appropiate plasmids, and purified on Prepac chromatography (BRL). The fragments were 5' end labelled by T4 polynucleotide kinase with [F'P]ATP (Amersham). MCI proteinMethanosarcina spp. CHTI 55 (DSM 2902) was grown as indicated in [14]. The protein MCI was prepared as previously described in [17] and purified as in [lo] with the m~ifications indicated in [15]. For~otion of the complexesComplexes were formed by addition of MC1 protein to DNA fragment in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing 50 mM NaCl. After equilibration for 45 min at 20°C samples were mixed with 115 volume of loading buffer (0.01% Bromophenol blue, 50% glycerol in TE). The ~on~ntrations of the protein and of the DNA were determined by absorption spectroscopy with an extinction coefficient of 1,lOOO M-'.crn-' per protomer at 280 nm and 1,300O M-'cm-' per bp at 260 nm, respectively. Gel electrophoresis of the complexesPolyacrylamide slab gels (7% acrylamide, 0.1% bisacrylamide) were cast in 45 mM Tris-boric acid, 1 mM EDTA (TBElt). Pre-run and run electrophoreses were performed at 10 V.cm-' for 1 and 3 h, respectively. The gels were dried and exposed to CGR films. The autoradiographs were analyzed by densitomet~ at 580 nm on a Camag Tic Scanner II densitometer. 335

Structure‐specific binding recognition of a methanogen chromosomal protein

“…This suggests that the structure of the junction bound to MC1 is neither that of the free junction in the absence of salt nor that of the free junction in the presence of MgCl 2. This hypothesis is consistent with the results of the experiments using KMnO 4 , which show that MC1 alters the structure of the fourway junction regardless of the presence or absence of divalent cations.…”

“…The results show that MC1 preferentially binds to four-way DNA junctions. Footprinting experiments using the hydroxyl radical and KMnO 4 show that MC1 is actually bound at the centre of the junction and suggest that the structure of the junction is pertubated by the binding of the protein.…”

“…CHTI 55 strain, MC1 is a small protein of 93 amino acids (11 kDa) that contains a high amount of charged residues (24% basic residues and 14% acidic residues) and no large hydrophobic domain [2]. With respect to its primary structure, MC1 has no similarity with any other eukaryotic, eubacterial or archaeal structural proteins [3].MC1 binds double-stranded DNA as a monomer in a noncooperative way with a binding site size of about 11 bp [4]. It protects DNA against thermal denaturation [5] and against radiolysis by fast neutrons [6].…”

“…MC1 binds double-stranded DNA as a monomer in a noncooperative way with a binding site size of about 11 bp [4]. It protects DNA against thermal denaturation [5] and against radiolysis by fast neutrons [6].…”

An archaeal SET domain protein exhibits distinct lysine methyltransferase activity towards DNA‐associated protein MC1‐α

“…Studies have shown that MC1 molecules associate with DNA in vivo, and are present at the ratio of 1 MC1 protein per 100 bp of DNA [26,27]. MC1 preferentially binds double‐stranded DNA as a monomer, and such interactions lead to DNA bending and negative supercoiling, thus protecting DNA against thermal denaturation [28–31]. In in vitro assays, addition of MC1 proteins has been shown to stimulate E. coli RNA polymerase activity at low protein to DNA ratios, but the RNA polymerase and subsequent transcription were inhibited at higher ratios [29].…”