Structural and Functional Analysis of a Bipolar Replication Terminus

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. 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. In many prokaryotic and some eukaryotic replicons, replication forks initiated at specific replication origins and moving bi-directionally are not terminated randomly but in regions delimited by polar replication termini (Ter).1 TheTer 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. TheTer 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 nonarresting 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 (1Bastia D. Mohanty B.K. DePamphilis M. DNA replication in Eukaryotic Cells.Cold Spring Harbor Laboratory Press. 1996; : 177-215Google Scholar, 2Bussiere D.E. Bastia D. Mol. Microbiol... 1999; 31: 1611-1618Google Scholar). Each Ter site of Bacillus subtilis binds to two interacting dimers of RTP to arrest forks in a polar mode (3Carrigan C.M. Pack R.A. Smith M.T. Wake R.G. J. Mol. Biol... 1991; 222: 197-207Google Scholar, 4Kaul S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 11143-11147Google Scholar). 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 (5Bussiere D.E. Bastia D. White S.W. Cell.. 1995; 80: 651-660Google Scholar). Second, what is the molecular mechanism of fork arrest (see reviews in Refs. 2Bussiere D.E. Bastia D. Mol. Microbiol... 1999; 31: 1611-1618Google Scholar and 6Wake R.G. King G. Structure.. 1997; 5: 1-5Google Scholar). 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 (7Meijer W.J.J. Smith M.T. Wake R.G. de Boer A.L. Venema G. Bron S. Mol. Microbiol... 1996; 19: 1295-1306Google Scholar) and comparing and contrasting the results with similar contacts derived from a normal unipolarTer 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 (8Ebright Y.W. Chen Y. Pendergrast P.S. Ebright R.H. Biochemistry.. 1992; 31: 10664-10670Google Scholar, 9Mazzarelli J.M. Ermacora M.R. Fox R.O. Grindley N.D. Biochemistry.. 1993; 32: 2979-2986Google Scholar, 10Pai K.S. Bussiere D.E. Wang F. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 10647-10652Google Scholar) and have determined the site-directed cleavage maps of the bipolar replication terminus. Furthermore, using the crystal structure of RTP (5Bussiere D.E. Bastia D. White S.W. Cell.. 1995; 80: 651-660Google Scholar) 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 mapping the points of arrest of the helicase and T7 RNA polymerase at each end of the Ter site and have determined the minimal 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. The pUC18-IRI (BS3) and pUC18-IRI (BS3) Rev. plasmids were constructed by transferring anXbaI-HindIII fragment that contained the IRI (unipolar Ter) from the pET22b-BS3 and pET22b-BS3 Rev. plasmids (11Mohanty B.K. Sahoo T. Bastia D. EMBO, J... 1996; 15: 2530-2539Google Scholar) into the pUC18 vector. The pUC18/19-Bipolar plasmids were constructed by cloning a 46-bp EcoRI-HindIII fragment containing the bipolar Ter site into the pUC18/19 vectors. The plasmids pET22b-IRI, (pET22b-BS3) and pET22b-IRI Rev. (pET22b-BS3 Rev) contained a unipolar Ter site in opposite orientations. The pET22b-Bipolar and pET22b-Bipolar Rev. plasmids were constructed by cloning an NdeI-HindIII and anNdeI-EcoRI fragment from pUC18-Bipolar and pUC19-Bipolar plasmids, respectively, in the pET22b vector. M13mp19-Bipolar and M13mp18-Bipolar clones were made by cloning a 46-bp DNA fragment containing the bipolar terminus into the M13mp18 and M13mp19 vectors, respectively. RTP contains a single and apparently solvent-inaccessible Cys at position 110 in the dimerization α-helix. No attempts were made to mutate this residue to a noncysteine moiety. Instead, additional Cys residues were introduced one at a time into the locations indicated (see Figs. 3 and 4) by site-directed mutagenesis (QuickChange kit, Stratagene), and the residues were derivatized with EPD and cleavage reactions were performed as published (8Ebright Y.W. Chen Y. Pendergrast P.S. Ebright R.H. Biochemistry.. 1992; 31: 10664-10670Google Scholar, 10Pai K.S. Bussiere D.E. Wang F. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 10647-10652Google Scholar). When DNA cleavage is catalyzed by an Fe–EDTA-conjugated DNA-binding protein, cleavage occurs at the C1′ and/or C4′ bonds of the sugar moieties within 3–4 Å from the location of the hydroxyradical generator, i.e. the iron atom. This iron atom, in turn, is located at a distance of 14 Å from the α-carbon atom of the a of the EPD the cleavage of the occurs at a distance of 14 3–4 Å from the α-carbon of the This a distance to be the locations of the on RTP and the cleavage sites on the DNA. The cleavage data were converted to distance constraints for with the distance constraints were converted to an for the molecular by as a in structure This not a the Fe–EDTA and the the is a way to the affinity cleavage data into distance constraints that be for of the The model of two dimers of RTP Å from the of the terminus DNA as a duplex of with the to the of each of the two subsites within the was to the protein-DNA there are that be to generate this the involved each structure of the protein and each sugar and base of the DNA as a that was of and The of the within such a are not to was to and the structure of the the of the within the the within the and of contact amino acid residues of RTP and nucleotide residues of the unipolar IRI and bipolar The unipolar terminus data are from K.S. Bussiere D.E. Wang F. White S.W. Bastia D. J... 1996; 15: Scholar). and to the two of one RTP and to the of the second contact points on the unipolar terminus. contact points on the bipolar the of the nucleotide a was to of the be that the by the are in and not the of an molecular the provide on how each binds its it is to specific contacts from the of and were as published T. Bastia D. Cell.. Scholar) with some This was as (11Mohanty B.K. Sahoo T. Bastia D. EMBO, J... 1996; 15: 2530-2539Google B.K. T. Bastia D. J. Scholar). have T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar, T. Mohanty B.K. Bastia D. J. 1995; Scholar). first goal was to a of the arrangement of two interacting dimers of RTP at the bipolar Ter a comparison of the bipolar Ter with a unipolarTer sequence showed at the sequence and at the sequence suggested that the of the bipolar Ter has sequence with and the have two sequences in a W.J.J. Smith M.T. Wake R.G. de Boer A.L. Venema G. Bron S. Mol. Microbiol... 1996; 19: 1295-1306Google Scholar, some of the base to amino acid contacts the two RTP dimers and the the protein was with DNA and with as T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; and the were and in The of the data is in data of the of the bipolar terminus is in of the contacts of the bipolar showed a symmetrical pattern as with that of the normal unipolar Ter site that showed contacts at its sequence than the site This result us to in the amino acid to nucleotide contacts RTP and the bipolar Ter site as one to the crystal structure of the complex to a of how the protein with DNA. the of complex that have to this time to further in and the of the structure we to the in We converted RTP to a site-directed chemical nuclease and it to generate an amino acid to nucleotide cleavage of the Ter complex. The structure of the with and the pattern of cleavage the iron atom contacts the as with the of the DNA are in The cleavage data were with to a three-dimensional model of the Ter complex. have for of DNA-protein interaction in and provide a to the of the crystal provide a of the protein-DNA complex that is by the crystal affinity the of a crystal to the of the nucleotide to amino contacts both in time and in (8Ebright Y.W. Chen Y. Pendergrast P.S. Ebright R.H. Biochemistry.. 1992; 31: 10664-10670Google Scholar). We affinity cleavage maps as constraints were from the cleavage data and were to generate a model of the Ter complex as in in a The residues and to the and the residues and from the and the were to and the were to The were to DNA that was at the to the The cleavage pattern of some is in are in the to RTP not DNA in the in the of RTP with EPD not DNA with RTP to generate that in DNA. be that the are to to the the from the location of the iron on which of the DNA double the iron atom residue a cleavage pattern that was to the consistent with the contact of the at the of Ter DNA The residues and both DNA at the 3 and This result is consistent with the fact that and are located only Å in the crystal structure of RTP (5Bussiere D.E. Bastia D. White S.W. Cell.. 1995; 80: 651-660Google Scholar). that the cleavage pattern was we the residue of the bipolar Ter by amino acid to a T. This on the bipolar Ter DNA the specific cleavage pattern to and a and cleavage pattern in the Ter the notion that the residue to contact was be that the contact is known to be necessary for binding of RTP to DNA T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar). of cleavage on both of DNA as with the cleavage of derivatized and in the published structure of the Ter K.S. Bussiere D.E. Wang F. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 10647-10652Google the residues and of each RTP with the of within each Ter and to the of the two dimers of RTP The of Ter complex is in The of both dimers are into the of the DNA the the and into the The are also for with the were are by crystal in the crystal structure and in that position in the The is by the of the and this a in the DNA structure by a of the This the normal base This in the DNA the of the RTP and at the of the there was only a of the DNA consistent with published results Wake R.G. King 1997; Scholar). The to the DNA were the rather than there within the interaction be investigated by one by Å the DNA are it is to that the bipolar terminus the bipolar of the replication it is of two of the sequence of this terminus that two and show a to the within the IRI (unipolar Ter) The critical in the are also in that each within the bipolar terminus is a of This binding arrangement a for the of this terminus. the pattern of contacts within both the subsites of the bipolar terminus is there are contacts residue and the DNA in the the subsites in the bipolar terminus. This is in to the IRI terminus residue of the for this in binding it show that RTP binds to unipolar and bipolar termini of has that two RTP dimers bind to the normal unipolar terminus in a T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar, Wake R.G. J. Mol. Biol... Scholar, Smith M.T. Wake R.G. Mol. Microbiol... 1993; Scholar). First, the affinity site is by one and the site is by the second We to the pattern of RTP binding to the bipolar terminus site with that of the unipolar site. The IRI fragment showed the in the of RTP with the first to of the site and with the second to of both the and site The fragment containing the bipolar terminus with RTP also showed a of its sites in a was (7Meijer W.J.J. Smith M.T. Wake R.G. de Boer A.L. Venema G. Bron S. Mol. Microbiol... 1996; 19: 1295-1306Google which be of of RTP dimers on the site. We to the bipolar terminus with to its to arrest replication forks in vitro and also to the points of arrest of the DNA. we have a in vitro replication of coli S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 11143-11147Google Scholar, T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar) to study the replication fork arrest by RTP to of the DnaB of subtilis have not by an replication that in of subtilis has not to We in this containing the IRI site in were in pUC18 plasmid and the bipolar terminus was in pUC18 and in with respect to origin of replication to generate pUC18-IRI and pUC19-Bipolar for study (see The plasmid DNA were in vitro using coli in the of of RTP and the were in a The pUC18-IRI Rev. containing the IRI site in with respect to the was in the of of RTP RTP not the replication fork in this the replication of the pUC18-IRI containing the IRI site in with respect to the replication in the of of RTP showed a with of and a and bp were We have RTP and have that at of the of the to the replication fork arrest in pUC18-IRI were of of of replication not The pUC18-Bipolar in the of and RTP over showed a than the and of the with of the of arrested and a of and replication also The pUC19-Bipolar was in the of and RTP in the of the pUC18-Bipolar the pUC19-Bipolar also showed replication arrest The than the of RTP the of and replication to be that the RTP to show in pUC18-Bipolar and pUC19-Bipolar was in comparison to that in pUC18-IRI and Rev. We the in vitro replication further to the nucleotide sequence replication was arrested in and pUC19-Bipolar The pUC18-IRI containing a site bp of the IRI site was with The pUC18-Bipolar and pUC19-Bipolar containing sites as in the pUC18/19 and were with The were in a with pUC18-Bipolar not the nucleotide sequence of the is arrested by the complex. The site the of the minimal effective sequence to arrest the helicase T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; T. Mohanty B.K. Bastia D. J. 1995; Scholar). the nucleotide sequences on both of the bipolar terminus replication fork is by the Ter complex. We have that RTP replication by the activity coli DnaB S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 11143-11147Google Scholar, T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar). We to the arrest of helicase in both of the bipolar terminus. M13mp18 and M13mp19 clones containing the IRI site have S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 11143-11147Google Scholar). The M13mp18-Bipolar and were to to generate the helicase and were for DNA unwinding activity of DnaB in the and of RTP. The were with a and data were as a in DnaB was arrested by RTP on the but not on the S. Mohanty B.K. Sahoo T. Patel I. Khan S. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 11143-11147Google T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar). was on the M13mp19-Bipolar as in in the of the M13mp18-Bipolar RTP by in with only an in the of Thus, the bipolar terminus replication and helicase activity from both one of the terminus the helicase than the other. We further investigated helicase by the bipolar terminus with using a by us T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar, T. Mohanty B.K. Bastia D. J. 1995; Scholar). The at the position was to the DNA and was by DNA polymerase in the of and one of reactions were each containing a of DnaB helicase activity J. we performed the helicase with only DnaB with DnaB in the of We by DnaB (or DnaB from the in the of of RTP. The M13mp18-Bipolar showed in DNA unwinding but not as as in the M13mp19-Bipolar consistent with the that one of the bipolar terminus was than the in the helicase. We to the effective sequence to arrest the helicase at the bipolar terminus in both orientations. reactions were with both the M13mp18-Bipolar and the M13mp19-Bipolar in the of each of the and and the were The DnaB unwinding activity was in the of of RTP to unwinding and was in and The of which there was a of of of was from the and and in a with the pUC18-Bipolar and the pUC19-Bipolar and The location of the critical in both of the bipolar terminus was determined from the and is in we have that the complex by and RNA in a polar fashion (11Mohanty B.K. Sahoo T. Bastia D. EMBO, J... 1996; 15: 2530-2539Google Scholar, B.K. T. Bastia D. J. Scholar). the bipolar terminus replication and DnaB helicase from both with we to and to what the also the arrest of The containing the bipolar terminus in were in pET22b plasmid as The clones containing IRI bipolar terminus were with and in the of RTP. RTP T7 RNA at IRI in a polar fashion (11Mohanty B.K. Sahoo T. Bastia D. EMBO, J... 1996; 15: 2530-2539Google Scholar). The pET22b-IRI (pET22b-BS3) in the of a single in the of a was with in the pET22b-IRI Rev. (pET22b-BS3 was in the of RTP When pET22b-Bipolar was in the of a was as in the of pET22b-Bipolar Rev. showed of a in the of RTP with less than the pET22b-Bipolar the of in of The blocking of IRI and bipolar were with a and are in and the data the The first part of the in this study was initiated with the of to the molecular basis of the polarity of arrest of the replication forks and the by comparing the nucleotide to amino acid contacts of RTP bound to the bipolar terminus with that of the unipolar terminus. We have also constructed a three-dimensional model of the Ter complex by the crystal structure of the (5Bussiere D.E. Bastia D. White S.W. Cell.. 1995; 80: 651-660Google Scholar) and the affinity cleavage data. The model that is consistent with mutagenesis and cross-linking data that suggested for the the and the of RTP in DNA binding K.S. Bussiere D.E. Wang F. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 10647-10652Google Scholar, K.S. Bussiere D.E. Wang F. White S.W. Bastia D. J... 1996; 15: Scholar). the to be the that the and the attempts to the and to generate affinity cleavage were of the bipolar Ter sequence of the plasmid pLS20 of subtilis not provide on the molecular basis of polarity. The data provide a of symmetrical contacts of RTP with the bipolar terminus but not amino acid to base contacts, which was from the affinity cleavage data. The affinity cleavage to of DNA-protein the crystal structure of the is has to be a to the DNA-protein derived by (8Ebright Y.W. Chen Y. Pendergrast P.S. Ebright R.H. Biochemistry.. 1992; 31: 10664-10670Google Scholar, 9Mazzarelli J.M. Ermacora M.R. Fox R.O. Grindley N.D. Biochemistry.. 1993; 32: 2979-2986Google Scholar, D. Structure.. 1996; Scholar). This study has revealed symmetrical base to amino acid contact in the bipolar as with the and contacts with the site and contacts with the site that were in the unipolarTer K.S. Bussiere D.E. Wang F. White S.W. Bastia D. Proc. Natl. Acad. Sci. U. S. A... 1996; 93: 10647-10652Google Scholar). This suggests that symmetrical RTP dimers to generate polarity by protein-DNA contacts in unipolar Ter in the the contacts polarity. In with RTP of the protein coli polar arrest of of the of the protein bound to a site T. 1996; Scholar). comparison of the Ter complex with that of the Ter complex also revealed that the contacts in the two are in some additional residue that is located in the of the two dimers contacts the part of the unipolarTer but contacts are not in the DNA-protein contact and by polarity by DNA binding of RTP to the and binding to the the mechanism also contact on one and contact at the We that DNA-protein interaction is one but not the only in polarity for the First, there is a of in vitro data that interaction in K.S. Bussiere D.E. White S.W. Bastia D. Cell.. 1996; Scholar). Second, we have that a structurally site located a replication of subtilis forks in a bipolar and D. J. in Scholar). The data the that protein-DNA interaction a that helicase to contact the contrahelicase of RTP. a symmetrical be one way of that is that but contacts at the and sites of a Ter such as was at the of subtilis generate a necessary for contact on both of the Ter of fork The that the binding of the two dimers of RTP to the bipolar Ter to show is consistent with that it is not to have two dimers of RTP binding to two sites to arrest a helicase but that the two dimers be T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar). be that the helicase arrest the minimal effective sequence to arrest the helicase from both not show the location the helicase The site at which of the DNA to be just the effective sequence for helicase In this it be to in that the of the helicase at the terminus not that of the helicase with the replication complex in vitro. it is interesting that replication termini also arrest RNA in a polar the not the notion that that on DNA DNA be arrested by RTP. T. Mohanty B.K. Bastia D. J. 1995; Scholar) showed that that replication are not arrested by RTP. The results of the helicase showed that RTP arrested DnaB helicase-catalyzed unwinding of DNA regardless of the of the from a to than one RTP helicase translocation rather than authentic it have to only the of of the rather than the T. Mohanty B.K. Patel I. Bastia D. EMBO, J... 1995; Scholar, J. 1992; Scholar). of unwinding of DNA regardless of the of the to be consistent with the that RTP arrested authentic DNA unwinding rather than just the translocation of the helicase. the of RTP to arrest RNA polymerase and suggest that RTP at site by binding to DNA. we have presented that is consistent with the notion that arrest not only interaction but also interaction the and the arrested K.S. Bussiere D.E. White S.W. Bastia D. Cell.. 1996; Scholar, and D. J. in Scholar). from with the protein of coli has also a critical role of the terminator interaction in fork J. and D. in that and RTP are structurally arrest the helicase by the same polar replication termini replication terminator protein base