Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.
The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.
Pai
K S
KS
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Bussiere
D E
DE
Wang
F
F
White
S W
SW
Bastia
D
D
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins; D001665:Binding Sites; D003196:Computer Graphics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000648:ultrastructure*; D003852:Deoxyribonucleoproteins / Q000648:ultrastructure; D004492:Edetic Acid / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D046911:Macromolecular Substances; C063112:rtP protein, Bacillus subtilis; D004492:Edetic Acid; D007501:Iron