Sequence-specific Interactions in the Tus-Ter Complex and the Effect of Base Pair Substitutions on Arrest of DNA Replication in Escherichia coli
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26448
ISSN1083-351X
AutoresFatma Filiz Coşkun-Ari, Thomas Hill,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences. Binding of Tus to aTer site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion. In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication.In vitro competition assays demonstrated that base pair substitutions at positions 8–19 had significant effects on the free energy of Tus binding (ΔΔG 0 of 1.5 to >4.0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo. Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Terinteractions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence. Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences. Binding of Tus to aTer site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion. In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication.In vitro competition assays demonstrated that base pair substitutions at positions 8–19 had significant effects on the free energy of Tus binding (ΔΔG 0 of 1.5 to >4.0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo. Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Terinteractions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence. The Tus protein of Escherichia coli binds to specific chromosomal sequences, called Ter sites, and arrests DNA replication in an orientation-dependent fashion (for review, see Ref. 1Hill T.M. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2. American Society for Microbiology, Washington, D. C.1996: 1602-1614Google Scholar). Replication forks approaching from the non-permissive side of the Tus-Ter complex are arrested, but replisomes approaching from the permissive side can pass through the complex. The Ter sites are oriented in the chromosome to permit DNA replication in the origin-to-terminus direction, but restrict replication forks traveling in the terminus-to-origin direction. Thus, the Tus-Ter complexes form a replication fork trap and prevent DNA replication forks from meeting in regions other than in the chromosomal terminus.The functional polarity demonstrated by Tus is reflected in the asymmetry of the protein-DNA complex, whose crystal structure has been recently solved (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar). Tus binds as a monomer (3Sista P.R. Hutchison C.A. Bastia D. Genes Dev. 1991; 5: 74-82Crossref PubMed Scopus (20) Google Scholar, 4Coskun-Ari F.F. Skokotas A. Moe G.R. Hill T.M. J. Biol. Chem. 1994; 269: 4027-4034Abstract Full Text PDF PubMed Google Scholar) and contacts both strands of the Ter site on the nonpermissive of the complex, but only a single strand on the permissive side (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar, 5Gottlieb P.A Wu S. Zhang X. Tecklenburg M. Kuempel P. Hill T.M. J. Biol. Chem. 1992; 267: 7434-7443Abstract Full Text PDF PubMed Google Scholar). TheTer site DNA is nestled into a cleft formed by the two primary domains of Tus (amino and carboxyl domains) and the interstrand β-sheets that connect the two domains (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar). The primary determinants of base pair recognition and binding are mediated by the main two interstrand β-sheets, which penetrate deeply into the major groove of the Ter site, making both polar and hydrophobic contacts with the bases. Binding is also enhanced by extensive contacts between Tus and the phosphates in the DNA backbone. A total of 42 amino acid residues stretched along the length of the protein make contacts with the DNA.Tus binds to the chromosomal Ter sites with a very high affinity. The K obs for Tus binding to theTerB site ranges between 3.4 × 10−13m and 7.5 × 10−13m, depending on the buffer conditions used (5Gottlieb P.A Wu S. Zhang X. Tecklenburg M. Kuempel P. Hill T.M. J. Biol. Chem. 1992; 267: 7434-7443Abstract Full Text PDF PubMed Google Scholar, 6Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. J. Biol. Chem. 1995; 270: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Half-lives (t½) of the protein-DNA complex were determined to be 550 to 149 min, respectively, in these studies. The high affinity of the Tus-Ter interaction in conjunction with the distribution of protein-DNA contacts has been used to suggest that Tus can arrest DNA replication by functioning as a clamp on the DNA and preventing the unwinding activity of the DnaB helicase (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar, 5Gottlieb P.A Wu S. Zhang X. Tecklenburg M. Kuempel P. Hill T.M. J. Biol. Chem. 1992; 267: 7434-7443Abstract Full Text PDF PubMed Google Scholar, 7Lee E.H. Kornberg A. Hidaka M. Kobayashi T. Horiuchi T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9104-9108Crossref PubMed Scopus (113) Google Scholar, 8Lee E.H. Kornberg A. J. Biol. Chem. 1992; 267: 8778-8784Abstract Full Text PDF PubMed Google Scholar). Alternately, protein-protein interactions between Tus and the DnaB helicase have been postulated to mediate replication arrest. This latter model is based upon the specificity of Tus function (9Khatri G.S. MacAllister T. Sista P.R. Bastia D. Cell. 1989; 59: 667-674Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 10Sahoo T. Mohanty B.K. Lobert M. Manna A.C. Bastia D. J. Biol. Chem. 1995; 270: 29138-29144Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), differential ability of Tus to halt helicase unwinding when presented with different templates (11Hiasa H. Marians K.J. J. Biol. Chem. 1992; 267: 11379-11385Abstract Full Text PDF PubMed Google Scholar), and mutational studies on Tus (6Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. J. Biol. Chem. 1995; 270: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 12Skokotas A. Wrobleski M. Hill T.M. J. Biol. Chem. 1994; 269: 20446-20455Abstract Full Text PDF PubMed Google Scholar).Ter sequences were originally identified as 22–23 base pairs in length, based on sequence identity betweenTerA, TerB, and TerC (13Hill T.M. Pelletier A.J. Tecklenburg M. Kuempel P.L. Cell. 1988; 55: 459-466Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 14Hidaka M. Akiyama M. Horiuchi T. Cell. 1988; 55: 467-475Abstract Full Text PDF PubMed Scopus (55) Google Scholar). As additional sites were identified both in the chromosome and in plasmid replicons, it became apparent that the essential conserved elements of the Ter site were an 11-base pair "core" sequence (positions 9–19) and an upstream G-C base pair at position 6 (Fig. 1). Nucleoside analogs have been used to partially map the determinants of Tus binding (15Duggan L.J. Hill T.M. Wu S. Garrison K. Zhang X. Gottlieb P.A. J. Biol. Chem. 1995; 270: 28049-28054Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Duggan L.J. Asmann P. Hill T.M. Gottlieb P.A. Biochemistry. 1996; 35: 15391-15396Crossref PubMed Scopus (6) Google Scholar) and it was shown that (i) the G residues at positions 10, 13, and 17 within the core sequence contributed both major and minor groove interactions, (ii) the conserved G residue at position 6 contributed little to the overall stability of the Tus-Ter complex, and (iii) hydrophobic interactions with thymine methyl groups occurred at positions 8, 9, 12, 14, 16, and 19.The solution of the co-crystal structure has identified specific interactions between Tus and the Ter site, but provides no information about the relative contribution of each contact to the free energy of binding. Likewise, the nucleotide analog studies can measure the energetic contributions of individual bases, but cannot determine the effect of natural base pair substitutions on binding. In addition, neither technique can address the role that specific protein-DNA interactions play in the ability of the Tus-Ter complex to arrest DNA replication. With this in mind, we undertook a systematic mutational analysis of the TerB site to determine which natural base pair substitutions affect Tus binding in vitroand what effect base pair substitutions have on replication arrest function in vivo.RESULTSSixteen different base pairs of the TerB site, between positions 6 and 21, were subjected to substitution analysis (Fig.1). It was possible to collect all three single base-substitutions at each position, except for positions 7 and 19, where only two substitutions were isolated. Therefore, a total of 46 single base pair-substituted Ter mutants were isolated and examined further using two different assays. An equilibrium competition assay was used to determine the binding affinity changes of Tus to the mutant Ter sites and a replication intermediate assay was performed to examine the ability of the mutant Tersequences to arrest DNA replication in vivo.Observed Equilibrium Dissociation Constants, (Kobs)The observed equilibrium dissociation constant (K obs) for each single base pair substitution was determined by measuring the ability of the mutant Tersites to compete with a 32P-labeled oligomeric wild-typeTerB site for Tus binding under equilibrium conditions. Because the competition assays utilized mutant Ter sites embedded in a plasmid, the possibility of nonspecific binding of Tus to the vector sequences of the plasmid was first estimated using the following equation (25Jen-Jacobson L. Lesser D.R. Kupiewski M.R. Nucleic Acids and Mol. Biol. 1991; 5: 142-170Google Scholar), Kapp=KS+NNSKNSEquation 4 where K app is the observed equilibrium association constant, K S is the true equilibrium association constant, K KS is the equilibrium association constant for nonspecific DNA (previously determined to be in the range of 2.4 × 10−7m for Tus binding to a 33-base pair non-Teroligomer), 1F. F. Coskun-Ari and T. M. Hill, unpublished results. andN NS is the number of nonspecific binding sites in the plasmid (approximately 300 in pSPORT1). Based on this equation, we would expect nonspecific binding to become problematic when the binding equilibrium constant exceeds 1 × 10−9m.Nonspecific binding of Tus to DNA was then empirically measured in a competition assay by mixing increasing concentrations of pSPORT1 DNA (without a Ter site) with a fixed amount of labeledTerB DNA and determining the decrease in binding to the labeled DNA. Only at DNA concentrations higher than 1 × 10−9m was significant nonspecific binding of Tus to pSPORT1 observed (Fig. 2). Consequently, for all subsequent competition experiments, competitor DNA concentrations were kept in the range of 1 × 10−9 to 1 × 10−13m. Additionally, to determine if the plasmid contained any other sequences that might affect Tus-Ter binding (such as the presence of pseudosites), unlabeled TerB oligomer and plasmid DNA containing the TerB site were individually assayed in competition reactions. The K obs for oligomericTerB was found to be almost identical to theK obs of a TerB site embedded in plasmid DNA (8 × 10−13m and 8.6 × 10−13m, respectively), indicating a lack of pseudosites or other nonspecific interactions with the plasmid DNA.Figure 2Determination of nonspecific binding of Tus to plasmid pSPORT1. Labeled wild-type TerB oligomer at a concentration of 1 × 10−12m was added to the indicated concentrations of unlabeled pSPORT1 or TerBoligomer and allowed to equilibrate with Tus protein (2.1 × 10−12m) in KG200 binding buffer. Following filtration of samples, the number of counts/min retained on the filters was determined. "cpm Blk" is the counts/min retained on filters in the absence of competitor DNA. ○, 33-merTerB; •, plasmid pSPORT1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To calculate the observed equilibrium dissociation constants, the data obtained from the competition assays were fitted to a binding curve using nonlinear regression analysis. The K obsfor all single base pair-substituted Ter sequences are listed in Table I and the data from these measurements are presented in Fig. 3. The relative affinity changes for Tus binding to the mutant Tersites were also calculated in terms of free energy changes (ΔΔG 0) for each base pair substitution.Table IValues in parenthesis indicate the number of independent experimentsWT BPPositionSubstitutionK obsΔΔG°Relative arrest activity10 −13 mkcal/molwt-TerB9 ± 1—1-aNot determined.95 ± 7 (5)G·C6A·T35 ± 40.8324 ± 7 (3)C·G16 ± 40.367 ± 4 (3)T·A31 ± 120.7633 ± 8 (3)T·A7A·T46 ± 60.9979 ± 4 (2)C·G———G·C13 ± 10.24102 ± 8 (2)A·T8C·G668 ± 132.571 ± 2 (2)G·C139 ± 41.6527 ± 8 (3)T·A952 ± 42.796 ± 1 (2)T·A9A·T232 ± 331.95—C·G209 ± 391.8921 ± 3 (3)G·C454 ± 142.3514 ± 4 (2)G·C10A·T≥7500≥4.00ND1-bND, relative arrest activity was not detectable.C·G≥7500≥4.00NDT·A≥7500≥4.00NDT·A11A·T428 ± 162.3113 ± 4 (3)C·G507 ± 222.4113 ± 7 (3)G·C1204 ± 1812.922 ± 3 (3)T·A12A·T≥7500≥4.00NDC·G≥7500≥4.00NDG·C≥7500≥4.00NDG·C13A·T≥7500≥4.00NDC·G≥7500≥4.00NDT·A≥7500≥4.00NDT·A14A·T≥7500≥4.00NDC·G≥7500≥4.00NDG·C≥7500>4.00NDA·T15C·G1622 ± 563.10NDG·C≥7500≥4.00NDT·A≥7500≥4.00NDA·T16C·G6994 ± 683.97NDG·C6015 ± 2803.88NDT·A≥7500≥4.00NDC·G17A·T≥7500≥4.00NDG·C≥7500≥4.00NDT·A154 ± 211.7119 ± 5 (5)T·A18A·T≥7500≥4.00NDC·G4561 ± 2533.71NDG·C538 ± 52.4515 ± 7 (3)A·T19C·G≥7500≥4.00NDG·C4335 ± 3153.68NDT·A———A·T20C·G69 ± 171.2350 ± 13 (2)G·C165 ± 91.7520 ± 2 (2)T·A29 ± 50.7233 ± 1 (2)A·T21C·G93 ± 181.4161 ± 5 (3)G·C39 ± 160.8961 ± 15 (3)T·A11 ± 10.15—1-a Not determined.1-b ND, relative arrest activity was not detectable. Open table in a new tab Figure 3Determination of the observable binding constants (K obs) of Tus for substitutions at positions 6–21. Labeled wild-type TerB oligomer at a concentration of 1 × 10−12m was added to the indicated concentrations of competitor pS/Ter plasmid DNA and allowed to equilibrate with Tus protein (2.1 × 10−12m) in KG200 buffer. Following filtration of samples, the number of counts/min retained on the filters was determined. The line through the data points is the best theoretical fit according to Equation 1. "cpm Blk" is the counts/min retained on filters in the absence of competitor DNA. ○, wild-type TerB; •, base pair substitution A·T; ▪, base pair substitution G·C; ▴, base pair substitution T·A; ♦, base pair substitution C·G.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The results indicate that the greatest energetic penalties (ΔΔG 0 > 4.0 kcal/mol) are exacted by substitutions located in the 11-base pair core region, at positions 10 and 12–19. The magnitude of the free energy change obtained for substitutions in this region demonstrates that these positions are absolutely critical for Tus binding. A second class of important base pairs, where all substitutions resulted in aΔΔG 0 of 1.5–3 kcal/mol, are located at the front of the Tus-Ter complex and include positions 8, 9, and 11. The third class of substitutions were located at the edges of the complex and included the highly conserved GC base pair at position 6, as well as bases 7, 20, and 21. All substitutions but one in this third class imposed energetic penalties of less than 1.5 kcal.Examination of in Vivo Replication Arrest Ability of Mutant Ter SitesThe mutant Ter sites were also assayed in vivo to determine the effect base pair substitutions had on the ability of the altered Tus-Ter complexes to arrest DNA replication. For this purpose, an assay was used that identifies replication intermediates arising from arrested DNA replication. First, plasmids containing the mutant Ter sites (pS/Ter) were transformed into the tus + strain TH463, which also contains a compatible internal control plasmid, pACtet. Cells were grown to mid-log phase and whole cell DNA was isolated under conditions that leave replicative intermediates intact. The extracted DNA samples were then digested with EcoRV, which cuts pS/Ter between the origin and the Ter site, and subjected to electrophoresis. If DNA replication was not arrested at the mutant Ter site, only a linear pS/Ter DNA was produced by EcoRV digestion. However, if DNA replication was arrested at the mutant Ter site, the EcoRV digest produced a double Y structure which migrates slower on an agarose gel than the linear fragment. Therefore, the appearance of a slower-migrating band indicated that the mutant Ter site still retained in vivo replication arrest activity. Replication intermediate and linear bands were visualized by Southern blot analysis using labeled pSPORT1 and pACtet as probes (Fig.4).Figure 4Replication arrest activities of selected substitutions in vivo. Whole cell DNA was isolated under nondenaturing conditions and digested with EcoRV prior to electrophoresis on a 0.8% agarose gel. The Southern blot of the gel was then probed with 32P-labeled pSPORT1 and pACtet plasmids to visualize the pS/Ter and the internal control plasmid pACtet. The linear pS/Ter band is 4.0 kilobases in length; arrest of DNA replication produces two slower migrating bands (∼6.5 kilobase) containing a single or double Y-fork.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In a previous study using the replication intermediate assay to examine mutant Tus proteins (6Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. J. Biol. Chem. 1995; 270: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), two factors were influenced by in vivo replication arrest activity. First, the ratio of the replication intermediate band to the linear band differed from one mutant to the other, depending on the efficiency of the Tus-Ter complex. Second, the total amount of plasmid DNA, which is related to the plasmid copy number, varied in each mutant as well. These factors were expected to vary in the replication intermediate assay because it was previously shown that the presence of a functional Ter site affected the copy number of pBR322-type plasmids (14Hidaka M. Akiyama M. Horiuchi T. Cell. 1988; 55: 467-475Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 26Bierne H. Ehrlich S.D. Michel B. J. Bacteriol. 1994; 176: 4165-4167Crossref PubMed Google Scholar) and also affected the accumulation of replication intermediates relative to the amount of linear plasmid (26Bierne H. Ehrlich S.D. Michel B. J. Bacteriol. 1994; 176: 4165-4167Crossref PubMed Google Scholar). Thus, to determine the efficiency of replication arrest in this previous study, the copy number of the plasmid and the ratio of replication intermediate to linear plasmid forms were considered when making quantitative comparisons between the mutant and wild-type Tus proteins.In this study, we also observed a general correlation between the efficiency of the Ter site and the plasmid copy number; however, we were unable to include this effect when quantitating the efficiency of replication arrest by the mutant Ter sites. This is because the correlation between the effectiveness of theTer sites and the copy number of the plasmids was not always consistent with the pSPORT1 vector. The most obvious inconsistency was observed when comparing the pSPORT1 vector without a Tersite and the same plasmid with the wild type TerB site: the copy number of the two plasmids were almost identical (1:1.3 ratio; Fig. 4). In previous studies (6Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. J. Biol. Chem. 1995; 270: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 26Bierne H. Ehrlich S.D. Michel B. J. Bacteriol. 1994; 176: 4165-4167Crossref PubMed Google Scholar) the copy of theTer-less plasmid was always significantly higher (twice as much) than the same plasmid with the TerB site. We also observed a significant increase in the plasmid copy number when a weakTer site was substituted for the TerB site. In several cases, such as substitutions at 12, 13, 14, and 15, the copy number of the Ter-containing plasmid was as much as four times higher than the copy number of the pSPORT1 alone (Fig.4). 2F. F. Coskun-Ari and T. M. Hill, unpublished data. At this point, we cannot explain these inconsistencies. Consequently, only the ratio of the Y-fork to linear band was used to quantitate Ter site efficiency in this study.We also noted two distinct replication intermediate bands in samples of functional Ter mutants and in the WT 3The abbreviation used is: WT, wild type. TerB. We speculate that the doublet replication intermediate bands are generated from head-to-tail pSPORT dimers, which were found in significant quantities in our plasmid preparations (data not shown). Dimeric plasmids can contain either one or two replication bubbles, depending upon whether initiation has occurred at one or both of the replication origins. Subsequent digestion of the dimeric plasmids withEcoRV would produce replication intermediates with either a single or double Y. These two DNA species would migrate differently in an agarose gel, presumably giving rise to the replication intermediate doublet. The appearance of doublet replication intermediate bands has been observed previously in studies of Tus mutants, in which plasmid pHV750T2+ was used to measure replication arrest function (6Skokotas A. Hiasa H. Marians K.J. O'Donnell L. Hill T.M. J. Biol. Chem. 1995; 270: 30941-30948Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Because the two bands in the doublet are believed to result from replication arrest, both bands were excised together when measuring the relative replication arrest activity of the substituted Tersites.A final observation regarding the in vivo assay was that the relative amounts of the two replication arrest bands varied in samples according to the strength of the Ter site (Fig. 4, position 6 mutants). In the WT TerB and the 6T·A mutant the banding pattern was the same: the slower-migrating band of the doublet, which was assumed to be the double Y form, was predominant over the faster-migrating band of the doublet, or the single Y-fork form. However, in the 6C·G mutant the single Y-fork form was found to be the most predominant band. Finally, in the 6A·T mutant sample the two Y-fork forms were present in equal amounts. The observation that the double Y was predominant in strong Ter sites and the single Y was predominant in weak Ter sites was consistent and reproducible, and seen with several other Ter mutants (positions 8, 9, and 18; Fig. 4). The reason for such a banding pattern is currently unknown.The efficiency of replication arrest in each of Ter mutant is presented in Table I. Base pair substitutions in the core region of the Ter site had the most significant effects on replication arrest activity and correlated with loss of DNA binding (Fig.5). In general,ΔΔG 0 values greater than 2.5 kcal completely abolished the ability of the Tus-Ter complex to halt DNA replication in vivo (Table I). Substitutions that resulted in low levels of replication arrest activity (from 20 to 1% of wild type levels) were typically associated withΔΔG 0 values of 1.7 to 2.5 kcal, with the notable exception of the 6C·G substitution, which had aΔΔG 0 value of only 0.35 kcal. Intermediate levels of replication arrest activity (80–20%) also showed a strong correlation with loss of binding activity, withΔΔG 0 values ranging from about 1 to 1.75. Again, notable deviations occurred with position 6 substitutions (A·T and T·A), and with the 20T·A substitution, all of which resulted inΔΔG 0 values of less than 1.Figure 5Correlation between loss of DNA binding and replication arrest activity. The ΔΔG 0value and replication arrest activity of base pair substitutions resulting in ΔΔG 0 values of less than 3.0 kcal/mol were plotted and a least squares analysis was used to fit a line to the data points (n = 16, r = 0.94). The dashed lines show one standard deviation from the calculated line. The values for 6A·T, 6C·G, 6T·A, and 20T·A, which were not included in the linear regression calculation, are also shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONA systematic mutational analysis of the E. coli Tersequence was undertaken to investigate the contribution of individual base pairs to the binding and function of the Tus protein. Substitutions were made at positions 6–21, which were identified previously as highly conserved bases associated with Tus binding (13Hill T.M. Pelletier A.J. Tecklenburg M. Kuempel P.L. Cell. 1988; 55: 459-466Abstract Full Text PDF PubMed Scopus (76) Google Scholar,14Hidaka M. Akiyama M. Horiuchi T. Cell. 1988; 55: 467-475Abstract Full Text PDF PubMed Scopus (55) Google Scholar). It is now known that position 5 is also contacted by Tus, through residue 198 (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar). However, this information was unavailable to us at the time this project was initiated and substitutions at this position were not investigated.Effects of Substitutions at Positions 6 and 7The G·C base pair at position 6 is conserved in all known Ter sites, however, the results reported here and in studies with nucleoside analogs (15Duggan L.J. Hill T.M. Wu S. Garrison K. Zhang X. Gottlieb P.A. J. Biol. Chem. 1995; 270: 28049-28054Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) suggest that this position contributes little to the stability of the Tus-Ter complex. The minimal change associated with the free energy of binding (ΔΔG 0 values of less than 1) may indicate that the Arg198 residue of Tus, which normally contacts the N-3 atom of G6 and the N-3 atom of A5 (Fig.6), remains anchored to position 5 when substitutions are made at position 6. Alternately, contact between Arg198 and the DNA could be completely lost when substitutions are made at position 6. In either scenario, it is possible that the position 6 substitutions do not have significant effects on Tus binding because the primary contacts for binding of this domain of Tus are made by other amino acids in the L3 loop and αVII domain (Ser193, Val200, and Trp208) with the phosphate group and deoxyribose moieties around base pair 6. With this in mind, it is of some interest to note that thetus gene of Salmonella has an alanine substituted for arginine at position 198, whereas the residues at positions 193, 200, and 208 are identical. 4A. Nilles and T. Hill, unpublished results. Figure 6Contacts between Tus andTer. Adapted from Kamada et al. (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar).Arrows show the sites of interaction between amino acid side chains and groups in the base pairs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Even though the effects of position 6 substitutions on DNA binding were minimal, the in vivo replication intermediate assay indicated a strong preference for the G·C base pair at this position. Replication arrest activities of the mutants were found to be significantly reduced (Fig. 4), with the C·G substitution showing the greatest effect (7% of the efficiency of the WT TerB). Furthermore, these substitutions showed the greatest deviance from the correlation between loss of DNA binding and loss of replication arrest activity (Fig. 5).In contrast to position 6, position 7 is not well conserved inTer sites (Fig. 7) and no amino acid residues of Tus contact this base pair (2Kamada K. Horiuchi T. Ohsumi K. Shimamoto Morikawa K. Nature. 1996; 383: 598-603Crossref PubMed Scopus (89) Google Scholar). The two substitutions made at position 7 did
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