Structural and functional analysis of a bipolar replication terminus. Implications for the origin of polarity of fork arrest.
We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter.RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.
Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
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D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D018741:Replication Origin; D013379:Substrate Specificity; D014764:Viral Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C063112:rtP protein, Bacillus subtilis; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004265:DNA Helicases; D053488:DnaB Helicases