No abstract

HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved "relic" DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a "HRS60-family". Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.

High-mobility group (HMG) 1 is a relatively highly abundant chromosomal protein with structuralrather than sequence-specific preference for binding to DNA. HMG1 has two highly related, folded domains A and B (HMG boxes), attached by a short basic region to an acidic C-terminal domain. We have studied binding of the B-domain of HMG1 protein and its mutants to supercoiled DNA by a gelretardation assay and electron microscopy. Using a gel-retardation assay, we have demonstrated that HMG1 or HMG1 lacking the acidic C-terminal domain [i.e. HMG1(AϩB) bi-domain], but not the isolated B-domain, could preferentially bind supercoiled over-relaxed closed circular or linear DNA. Mutational analysis of the HMG1 B-domain revealed that replacement of Lys96 of the extended N-terminal segment (and much less the neighboring Arg97) and Lys128 of helix II to glutamic acid severely impaired binding of the HMG box domain to supercoiled DNA. The latter mutation within helix II significantly decreased the A-helical content of the B-domain as revealed by circular dichroism. We have also shown that mutation of several residues within helix I of the B-domain, in particular Arg110, resulted in a diminished binding to supercoiled DNA as revealed by intensive smearing and reduced retardation of the protein/DNA complexes. These findings indicated that the extended N-terminus, helix I and helix II of the HMG1 B-domain are likely in contact with DNA. Electron microscopy revealed that the B-domain could bind to supercoiled DNA at higher HMG/DNA molar ratios as oligomeric protein beads with subsequent association of the beads into large nucleoprotein complexes from which many looped DNA molecules emerged. Most of the introduced mutations within all three helices of the B-domain (involving mainly basic and aromatic residues) abolished formation of the large nucleoprotein complexes even though the binding of the HMG box to supercoiled DNA was retained as revealed by a gel-retardation assay. A model for the interaction of the B-domain of HMG 1 with supercoiled DNA is presented and discussed.Keywords : high-mobility-group protein 1 (HMG1) ; high-mobility-group box; supercoiled DNA; gel retardation; electron microscopy.High-mobility-group (HMG) proteins 1 and 2 are evolution-HMG box, like the A-domain HMG box, consists of the extended N-terminal strand and three helical segments linked by ary highly conserved and abundant non-histone chromatin-associated proteins, implicated in transcription, replication, and re-short turns, and is arranged in a remarkable flat and distinct arrowhead V-or L-shaped form. combination in vitro [1], as well as in chromatin organization [28].The two HMG box domains of HMG1 apparently lack DNA-sequence specificity but are able to recognize different Mammalian HMG1 has two related but distinct repeats of a 70Ϫ80 amino acid sequence (folded DNA-binding domains A DNA structures. HMG1 and its isolated HMG box domains preferentially bind to bent, kinked, and unwound DNA such as cisand B), HMG boxes [2], attached by a short basic...

Electron microscopy has shown that non-histone chromosomal HMGl could induce DNA looping or compaction in the presence (but not in the absence) of Ca". The effect of calcium on DNA looping and compaction was interpreted as calcium binding to the acidic C-domain of HMGl. Both individual DNA-binding HMGl-box domains A and B were found to be involved in DNA looping and compaction. Treatment of HMGl with a thiol-specific reagent, N-ethylmaleimide, inhibited the ability of the protein to induce DNA looping and compaction but not the electrostatic interaction with DNA. These results indicated that cysteine-sulfhydryl groups of the HMGl-box domains A and B are specifically involved in DNA looping and compaction, and that in the absence of calcium the acidic C-domain down-regulates these effects by modulation of the DNA-binding properties of the HMGl-box domains.