The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif

Ultrastructure of interphase chromosomes

A study of the ultrastructure of interphase chromosomes of frog erythrocytes and human leucocytes suggested that the 500, 250 and 100 Å microfibrils observed in different preparations may represent different orders of coiling of a basic 40-60 Å microfibril. Unlike the situation in lampbrush chromosomes, DNAse digestion did not result in the disruption of microfibril continuity. It seems likely that DNA in a loosely bound DNA-histone complex may be more prone to fragmentation than in a more tightly bound complex. The uncoiling of microfibrils brought about by treatments with 1 M ammonium acetate and trypsin supports Zubay's [23] model involving histones in the supercoiling of DNA

Conformational changes in a replication origin induced by an initiator protein

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins α, β, and γ. Binding of the initiator to the direct repeats induced a marked bending of the region of γ replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin

Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein

Initiation of DNA replication from on β of plasmid R6K requires the presence of the on γ sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in γ increases binding of the protein at the very weak binding site present in on β by cooperativity at a distance. The γ-β interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated γ-β interaction in vitro suggests that the cooperative interaction of γ-bound protein with the β sequence by DNA looping is an early step in the initiation of DNA replication at the β origin of R6K