Structural and Functional Analysis of a Bipolar Replication Terminus IMPLICATIONS FOR THE ORIGIN OF POLARITY OF FORK ARREST* Received for publication, December 4, 2000 Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M010940200 Bidyut K. Mohanty‡, Dirksen E. Bussiere§, Trilochan Sahoo¶, Karnire S. Pai‡, Wilfried J. J. Meijer, Sierd Bron**, and Deepak Bastia‡ ‡‡ From the ‡Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, the §Department of Biological Chemistry, Structural Biology Group, Chiron Corporation, Emeryville, California 94608, the ¶Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, the Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma, Canto Blanco, 28049 Madrid, Spain, and the **Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Haren, The Netherlands We have delineated the amino acid to nucleotide con- tacts 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 termi- nus suggests that the DNA-protein contacts play an im- portant 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 fea- tures of the bipolar TerRTP complex derived from mu- tational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB he- licase and T7 RNA polymerase in vitro in both orienta- tions. 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. In many prokaryotic and some eukaryotic replicons, replica- tion forks initiated at specific replication origins and moving bi-directionally are not terminated randomly but in regions delimited by polar replication termini (Ter). 1 The Ter sites are usually short sequences that specifically bind to a replication terminator protein (RTP) and arrest fork moving in only one direction with respect to the origin but not the other. The Ter sites are usually located in two clusters of opposite polarity in such a way that the forks moving clockwise on a circular chromosome pass through the first cluster (that has the non- arresting polarity) and are arrested at the termini of the second cluster, which has blocking polarity. The same is true for forks moving in a counterclockwise direction (1, 2). Each Ter site of Bacillus subtilis binds to two interacting dimers of RTP to arrest forks in a polar mode (3, 4). Thus, there are two interesting mechanistic questions to be addressed in this context. First, how is the polarity of fork arrest generated, considering the fact that the dimeric RTP has a symmetrical structure (5). Second, what is the molecular mechanism of fork arrest (see reviews in Refs. 2 and 6). In this study we have endeavored to address the first question. We have approached the question by determining the amino acid to nucleotide contacts of RTP bound to the novel naturally occurring bipolar terminus of the plasmid pLS20 (7) and com- paring and contrasting the results with similar contacts de- rived from a normal unipolar Ter site. We have converted RTP to a site-directed chemical nuclease by coupling it to an organic Fe–EDTA conjugate at certain rationally selected critical amino acid residues that are known to be involved in contacting Ter DNA (8 –10) and have determined the site-directed cleav- age maps of the bipolar replication terminus. Furthermore, using the crystal structure of RTP (5) and the cleavage maps mentioned above, we have constructed a model of the two dimers of RTP bound to the bipolar Ter. The model revealed a relatively symmetrical amino acid to nucleotide contact pattern that presumably contributed to nearly equal binding affinity of both subsites of the bipolar Ter to RTP. The results suggest that the pattern of protein-DNA interaction at the Ter sites is probably one of the parameters that generate polarity (or the lack of it) at the Ter sites. In the second part of this study, we have investigated the biochemical properties of the bipolar Ter with the goal of map- ping the points of arrest of the helicase and T7 RNA polymer- ase at each end of the Ter site and have determined the mini- mal effective sequence necessary to arrest the helicase. We have determined an “activity footprint” of the contrahelicase activity of RTP on the bipolar Ter that showed that RTP was able to arrest helicase-catalyzed unwinding of double stranded DNA in a length-independent fashion over a range of a less than a 100 bp to over 1500 bp. This result is consistent with the notion that RTP arrested authentic DNA unwinding and not just helicase translocation. Thus, the data presented provide not only some insight into the molecular basis of the origin of polarity but also the characteristics of the bipolar terminus in vitro. * This work was supported by a grant from NIGMS and a merit award from NIAID of the National Institutes of Health (to D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡‡ To whom correspondence should be addressed. Tel.: 919-684-3521; Fax: 919-684-8735; E-mail: basti002@mc.duke.edu. 1 The abbreviations used are: Ter, polar replication termini; RTP, replication terminator protein; bp, base pairs, EPD, (S)-(2-pyridyl-thio) cysteaminyl-EDTA; IRI, inverted repeat I. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 16, Issue of April 20, pp. 13160 –13168, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 13160 by guest on May 23, 2020 http://www.jbc.org/ Downloaded from