Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.
The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.
Sahoo
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Department of Microbiology, Duke University Medical Center, Durham, NC 27710.
Mohanty
B K
BK
Patel
I
I
Bastia
D
D
D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004265:DNA Helicases / Q000037:antagonists & inhibitors; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; C063112:rtP protein, Bacillus subtilis; D004265:DNA Helicases; D053488:DnaB Helicases