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Jen-Jacobson, L., and L.A. Jacobson (2008) The role of water and the effects of small ions in site-specific protein-DNA interactions. Pp in Structural Biology of Protein-Nucleic Acid Interactions, Rice, P.A., and C. Correll, Ed. Royal Society of Chemistry Publishing, Cambridge Water and small ions play important or even preeminent roles in determining the specificity and thermodynamic properties of protein-DNA interactions. These influences are exerted not only by water or ionic ligands that are released from the macromolecules upon protein-DNA binding, but also by water molecules, and perhaps small ions, that remain bound to the protein-DNA complexes. The release of water from nonpolar surfaces to bulk solvent (hydrophobic effect) is likely the largest single energetic factor that drives site-specific protein-DNA binding. This bulk solvent-release process cannot itself be a determinant of sequence specificity, yet paradoxically enhances specificity because it does not occur unless sequence-specific interactions bring complementary protein and DNA surfaces into sufficiently intimate apposition to expel water from the interface. If we are to aspire to a quantitative rather than qualitative understanding of the roles of water and small ions in macromolecular processes, we will ultimately be compelled to seek deeper knowledge of fundamental physicochemical issues, such as the detailed structure and dynamics of water surrounding macromolecules, the hydration of small ions, and the dynamic behavior of proteins, DNA and their complexes in solution. Sapienza, P.J., J.M. Rosenberg, and L. Jen-Jacobson (2007) Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease. Structure 15:1368-1382 Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI( *)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI( *) sites that structurally resemble the specific wild-type complex with GAATTC. Sapienza, P.J., C.A. Dela Torre, W.H. .4.t. McCoy, S.V. Jana, and L. Jen-Jacobson (2005) Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases. J. Mol. Biol. 348:307-324 Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations. Kurpiewski, M.R., L.E. Engler, L.A. Wozniak, A. Kobylanska, M. Koziolkiewicz, W.J. Stec, and L. Jen-Jacobson (2004) Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease. Structure 12:1775-1788 Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other. Shuttleworth, G., M.J. Fogg, M.P. Kurpiewski, L. Jen-Jacobson, and B.A. Connolly (2004) Recognition of the pro-mutagenic base uracil by family B DNApolymerases from Archaea. J. Mol. Biol. 337:621-634 Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer#templates that contain uracil four bases in front of the primer#template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer#templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation. Engler, L.E., P. Sapienza, L.F. Dorner, R. Kucera, I. Schildkraut, and L. Jen-Jacobson (2001) The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC. J. Mol. Biol. 307:619-636 The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg2+) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the best (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg2+, first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca2+ at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage. Connolly, B.A., H.H. Liu, D. Parry, L.E. Engler, M.R. Kurpiewski, and L. Jen-Jacobson (2001) Assay of restriction endonucleases using oligonucleotides. Methods Mol. Biol. 148:465-490 Watrob, H., W. Liu, Y. Chen, S.G. Bartlett, L. Jen-Jacobson, and M.D. Barkley (2001) Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor. Biochemistry 23:683-692 EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 Å in apoenzyme, decreasing to 26 Å in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg2+ were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures. Jen-Jacobson, L., L.E. Engler, J.T. Ames, M.R. Kurpiewski, and A. Grigorescu (2000) Thermodynamic parameters of specific and nonspecific protein-DNA binding. Supramol. Chem. 12:143-160 Proteins that bind preferentially to specific recognition sites on DNA also bind more weakly to nonspecific DNA. We have studied both specific and non-specific binding of the EcoRI and BamHI restriction endonucleases, and determined enthalpic and entropic contributions to binding free energy (DG°bind) using both the van't Hoff method and isothermal titration calorimetry. Specific binding is characterized by a strongly negative DC°p and can be either enthalpy-driven or entropy-driven, depending on temperature. Nonspecific binding has a DC°p of approximately zero and is enthalpy-driven. A strongly negative DC°p is the "thermodynamic signature" of site-specific binding, because it reflects the characteristics of a tight complementary recognition interface: the burial of previously hydrated nonpolar surface and restriction of configurational-vibrational freedoms of protein, DNA, and water molecules trapped at the protein-DNA interface. These factors are absent in nonspecific complexes. We probed the contributions to DC°p by varying the sequence context surrounding the recognition site. As DG°bind improves, DC°p, DH° and DS° all become more negative, and there is a linear correlation between DH° and DS° (enthalpy-entropy compensation). Because these context variations do not change the protein-base or protein-phosphate contacts, the hydrophobic contribution or the number of trapped water molecules at the interface, we conclude that a better sequence context improves the "goodness of fit" in the interface and and thus increases the magnitude of the negative configurational-vibrational contribution to DC°p. Jen-Jacobson, L., L. Engler, and L.A. Jacobson (2000) Structural and thermodynamic strategies for site-specific DNA binding proteins. Structure Fold Des. 8:1015-1023 Background: Site-specific protein-DNA complexes vary greatly in structural properties and in the thermodynamic strategy for achieving an appropriate binding free energy. A better understanding of the structural and energetic engineering principles might lead to rational methods for modification or design of such proteins. Results: A novel analysis of ten site-specific protein-DNA complexes reveals a striking correspondence between the degree of imposed DNA distortion and the thermodynamic parameters of each system. For complexes with relatively undistorted DNA, favorable enthalpy change drives unfavorable entropy change, whereas for complexes with highly distorted DNA, unfavorable DH° is driven by favorable DS°. We show for the first time that protein-DNA associations have isothermal enthalpy-entropy compensation, distinct from temperature-dependent compensation, so DH° and DS° do not vary independently. All complexes have favorable DH° from direct protein-DNA recognition interactions and favorable DS° from water release. Systems that strongly distort the DNA nevertheless have net unfavorable DH° as the result of molecular strain, primarily associated with the base pair destacking. These systems have little coupled protein folding and the strained interface suffers less immobilization, so DS° is net favorable. By contrast, systems with little DNA distortion have net favorable DH°, which must be counterbalanced by net unfavorable DS°, derived from loss of vibrational entropy (a result of isothermal enthalpy-entropy compensation) and from coupling between DNA binding and protein folding. Conclusions: Isothermal enthalpy-entropy compensation implies that a structurally optimal, unstrained fit is achieved only at the cost of entropically unfavorable immobilization, whereas an enthalpically weaker, strained interface entails smaller entropic penalties. Malygin, E.G., V.V. Zinoviev, N.A. Petrov, A.A. Evokimov, L. Jen-Jacobson, V.G. Kossykh, and S. Hattman (1999) Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase. Nucleic Acids Res. 27:1135-1144 The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on Km(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).
Liu, W., Y. Chen, H. Watrob, S.G. Bartlett, L. Jen-Jacobson, and M.D. Barkley (1998) N-termini of EcoRI restriction endonuclease dimer are in close proximity on the protein surface. Biochemistry 37:15457-15465 The N-terminal region of EcoRI endonuclease is essential for cleavage yet is invisible in the 2.5 Å crystal structure of endonuclease-DNA complex [Kim, Y., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990) Science 249, 1307-1309]. We used site-directed fluorescence spectroscopy and chemical cross-linking to locate the N-terminal region and assess its flexibility in the absence and presence of DNA substrate. The second amino acid in each subunit of the homodimer was replaced with cysteine and labeled with pyrene or reacted with bifunctional cross-linkers. The broad absorption spectra and characteristic excimer emission bands of pyrene-labeled muteins indicated stacking of the two pyrene rings in the homodimer. Proximity of N-terminal cysteines was confirmed by disulfide bond formation and chemical cross-linking. The dynamics of the N-terminal region were determined from time-resolved emission anisotropy measurements. The anisotropy decay had two components: a fast component with rotational correlation time of 0.3-3 ns representing probe internal motions and a slow component with 50-100 ns correlation time representing overall tumbling of the protein conjugate. We conclude that the N-termini are close together at the dimer interface with limited flexibility. Binding of Mg2+ cofactor or DNA substrate did not affect the location or flexibility of the N-terminal region as sensed by pyrene fluorescence and cross-linking, indicating that substrate binding is not accompanied by folding or unfolding of the N-terminus. Jen-Jacobson, L. (1997) Protein-DNA recognition complexes: Conservation of structure and binding energy in the transition state. Biopolymers 44:153-180 Jen-Jacobson, L. (1997) Protein-DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 244:153-180 This paper considers how enzymes that catalyze reactions at specific DNA sites have been engineered to overcome the problem of competitive inhibition by excess nonspecific binding sites on DNA. The formation of a specific protein-DNA recognition complex is discussed from both structural and thermodynamic perspectives, and contrasted with formation of nonspecific complexes. Evidence (from EcoRI and BamHI endonucleases) is presented that a wide variety of perturbations of the DNA substrate alter binding free energy but do not affect the free energy of activation for the chemical step; that is, many energetic factors contribute equally to the recognition complex and the transition-state complex. This implies that the specific recognition complex bears a close resemblance to the transition-state complex, such that very tight binding to the recognition site on the DNA substrate does not inhibit catalysis, but instead provides energy that is efficiently utilized along the path to the transition state. It is suggested that this view can be usefully extended to "noncatalytic" site-specific DNA-binding proteins like transcriptional activators and general transcription factors. Engler, L.E., K.K. Welch, and L. Jen-Jacobson (1997) Specific binding by EcoRV endonuclease to its DNA recognition site. J. Mol. Biol. 269:82-101 Restriction endonuclease EcoRV has been reported to be unable to distinguish its specific DNA site, GATATC, from non-specific DNA sites in the absence of the catalytic cofactor Mg2+, and thus to exercise sequence specificity solely in the catalytic step. In contrast, we show here that under appropriate conditions of pH and salt concentration, specific complexes with oligonucleotides containing the GATATC site can be detected by either filter-binding or gel-retardation. Equilibrium binding constants (K(A)) are easily measured by both direct equilibrium and equilibrium-competition methods. The preference for "specific" over "non-specific" binding at pH 7 in the absence of divalent cations is about 1000-fold (per mole of oligonucleotide) or 12,000-fold (per mole of binding sites). Ethylation-interference footprinting shows that the "specific" complex includes strong contacts to the phosphate groups GpApTpApTC. Specific DNA binding is strongly pH-dependent, decreasing about 15-fold for each increase of one pH unit above pH 6, but non-specific binding is not; thus, binding specificity decreases with increasing pH. Gel retardation and filter-binding at pH < or = 7 yield essentially identical values of K(A) for specific-site binding, but at pH > 7 gel retardation significantly underestimates K(A). Specific-site binding is stimulated about 700-fold by Ca2+ (not a cofactor for cleavage), but with non-cleavable 3'-phosphorothiolate and 4'-thiodeoxyribose derivatives whose response to Ca2+ is similar to that of the parent oligonucleotide, Mg2+ stimulates binding only fourfold and twofold, respectively. Thus, binding specificity is not dramatically enhanced by Mg2+. Taking into account discrimination in binding and in the first-order rate constant for phosphodiester bond scission, the overall discrimination exercised against the incorrect site GTTATC is about 107-fold. EcoRV endonuclease is thus not a "new paradigm" for site-specific interaction without binding specificity, but like other type II restriction endonucleases achieves sequence specificity by discriminating both in DNA binding and in catalysis. Kurpiewski, M.R., M. Koziolkoewicz, A.L. Wilk, W. Stec, and L. Jen-Jacobson (1996) Chiral phosphorothioates as probes of protein interactions with individual DNA phosphoryl oxygens: essential interactions of EcoRI endonuclease with the phosphate at pGAATT. Biochemistry 35:8846-8864 The contact between EcoRI endonuclease and the "primary clamp" phosphate of its recognition site pGAATTC is absolutely required for recognition of the canonical and all variant DNA sites. We have probed this contact using oligonucleotides containing single stereospecific (Rp)- or (Sp)- phosphorothioates (Ps). At the GAApTTC position, where the endonuclease interacts with only one phosphoryl oxygen at the central DNA kink, Rp-Ps inhibits and Sp-Ps stimulates binding and cleavage [Lesser et al. (1992) J. Biol. Chem. 267, 24810-24818]: in contrast, at the pGAATTC position both diastereomers inhibit binding. For single-strand substitution, the penalty in binding free energy (DDG°bind) is slightly greater for Sp-Ps (+ 0.9 kcal/mol) than for Rp-Ps (+ 0.7 kcal/mol). Binding penalties are approximately additive for double-strand substitution (Rp,Rp-Ps or Sp,Sp-Ps). Neither Ps diastereomer in one DNA strand affects the first-order rate constants for cleavage in the unmodified DNA strand, and only Sp-Ps inhibits the cleavage rate constant (3-fold) in the modified DNA strand. Thus, the second-order cleavage rate (including binding and catalysis) is inhibited 14-fold by Sp-Ps and 45-fold by Sp,Sp-Ps. In the canonical complex, the phosphate at pGAATTC is completely surrounded by protein and each nonbridging phosphoryl oxygen receives two hydrogen bonds from the endonuclease, such that in either orientation the increased bond length of P-S- inhibits binding. However, the pro-Sp oxygen interacts with residues that are connected (by proximity or inter-side-chain hydrogen bonding) to side chains with essential roles in catalysis, so cleavage is preferentially inhibited when these side chains are slightly displaced by the Sp-Ps diastereomer. Jen-Jacobson, L., L.E. Engler, D.R. Lesser, M.R. Kurpiewski, and B. McVerry (1996) Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites. EMBO J. 15:2870-2882 We have studied the interaction of EcoRI endonuclease with oligonucleotides containing GAATTC sites bearing one or two adenine-N6-methyl groups, which would be in steric conflict with key protein side chains involved in recognition and/or catalysis in the canonical complex. Single-strand methylation of either adenine produces small penalties in binding free energy (DDG°(S) approximately +1.4 kcal/mol), but elicits asymmetric structural adaptations in the complex, such that cleavage rate constants are strongly inhibited and unequal in the two DNA strands. The dependences of cleavage rate constants on the concentration of the Mg2+ cofactor are unaltered. When either adenine is methylated on both DNA strands, DDG°(S) (approximately +4 kcal/mol) is larger than the expected sum of the DDG°(S) values for the single-strand methylations, because the asymmetric adaptations cannot occur. Cleavage rate constants are reduced by 600 000-fold for the biologically relevant GAmATTC/CTTmAAG site, but the GmAATTC/CTTAmAG site forms only a non-specific complex that cannot be cleaved. These observations provide a detailed thermodynamic and kinetic explanation of how single-strand and double-strand methylation protect against endonuclease cleavage in vivo. We propose that non-additive effects on binding and structural 'adaptations' are important in understanding how DNA methylation modulates the biological activities of non-catalytic DNA binding proteins.
Jen-Jacobson, L. (1995) Structural-perturbation approaches to thermodynamics of site-specific protein-DNA interactions. Methods Enzymol. 259:305-344 Lesser, D.R., M.R. Kurpiewski, T. Waters, B.A. Connolly, and L. Jen-Jacobson (1993) Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition. Proc. Natl. Acad. Sci., USA 90:7548-7552 We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an interstrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.
Lesser, D.R., A. Grajkowski, M.R. Kurpiewski, M. Koziolkiewicz, W.J. Stec, and L. Jen-Jacobson (1992) Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex. J. Biol. Chem. 267:24810-24818 We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.
Jen-Jacobson, L., D.R. Lesser, and M.R. Kurpiewski (1991) DNA eequence discrimination by EcoRI endonuclease. Nucleic Acids Mol. Biol. :141-170 Lesser, D.R., M.R. Kurpiewski, and L. Jen-Jacobson (1990) The energetic basis of specificity in the EcoRI endonuclease-DNA interaction. Science 250:725-872 High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by EcoRI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. EcoRI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence. Becker, M.M., D. Lesser, M. Kurpiewski, A. Baranger, and L. Jen-Jacobson (1988) "Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex. Proc. Natl. Acad. Sci., USA 85:6247-6251 The "UV footprinting" technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.
Jacobson, L.A., L. Jen-Jacobson, J.M. Hawdon, G.P. Owens, M.A. Bolanowski, S.W. Emmons, M.V. Shah, R.A. Pollock, and D.S. Conklin (1988) Identification of a putative structural gene for cathepsin D in Caenorhabditis elegans. Genetics 119:355-363 Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.
Sarkis, G.J., M.R. Kurpiewski, J.D. Ashcom, L. Jen-Jacobson, and L.A. Jacobson (1988) Proteases of the nematode Caenorhabditis elegans. Arch. Biochem. Biophys. 261:80-90 Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate. Jen-Jacobson, L., D. Lesser, and M. Kurpiewski (1986) The enfolding arms of EcoRI endonuclease: role in DNA binding and cleavage. Cell 23:619-629 The N-terminal segments of the EcoRI endonuclease dimer form part of mobile "arms" that encircle DNA in the recognition complex. By treating endonuclease-TCGCGAATTCGCG complexes with proteases, we have prepared a series of deletion derivatives lacking defined segments of the N-terminal region. The 5-12 segment is essential for DNA cleavage and forms one electrostatic interaction (per subunit) with DNA phosphate. These ionic contacts are directly across the double helix from the scissile phosphodiester bonds; they thus may permit the enfolding arms to immobilize DNA in apposition to the catalytic cleft and/or contribute to the unusual "kinked" conformation of DNA in the complex. Sequence specificity is fully retained when 28 residues are deleted from the N-terminus, but the complexes dissociate more rapidly. Jen-Jacobson, L., D. Lesser, and M. Kurpewski (1985) Functional role of the N-terminal domain of EcoRI endonuclease. J. Cell. Biochem. Suppl. 9:105 Jacobson, L.A., L. Jen-Jacobson, and A.P. Wnek (1985) The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli. Arch. Biochem. Biophys. 15:118-131 The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site. Frederick, C.A., J. Grable, M. Melia, C. Samudzi, L. Jen-Jacobson, B.C. Wang, P. Greene, H.W. Boyer, and J.M. Rosenberg (1984) Kinked DNA in crystalline complex with EcoRI endonuclease. Nature 309:327-331 The 3 A electron density map of a co-crystalline recognition complex between EcoRI endonuclease and the oligonucleotide TCGCGAATTCGCG reveals that a tight, complementary interface between the enzyme and the major groove of the DNA is the major determinant of sequence specificity. The DNA contains a torsional kink and other departures from the B conformation which unwind the DNA and thereby widen the major groove in the recognition site. Grable, J., C.A. Frederick, C. Samudski, L. Jen-Jacobson, D. Lesser, P. Greene, H.W. Boyer, K. Itakura, and J.M. Rosenberg (1984) Two-fold symmetry of crystalline DNA-EcoRI endonuclease recognition complexes. J. Biomol. Struct. Dyn. 1:1149-1160 Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding VM values of 3.1 A3/dal and 2.8 A3/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice. Jen-Jacobson, L., M. Kurpiewski, D. Lesser, J. Grable, H.W. Boyer, J.M. Rosenberg, and P.J. Greene (1983) Coordinate ion pair formation between EcoRI endonuclease and DNA. J. Biol. Chem. 258:14638-14646 The free energy of the binding reaction between EcoRI restriction endonuclease and a specific cognate dodecadeoxynucleotide (d(CGCGAATTCGCG)) has contributions from both electrostatic and nonelectrostatic components. These contributions were dissected by measuring the effects of varying salt concentration on the equilibrium binding constant and applying the thermodynamic analyses of Record et al. (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158). Endonuclease mutation S187 (Arg 187 to Ser) (Greene, P. J., Gupta, M., Boyer, H. W., Brown, W. E., and Rosenberg, J. M. (1981) J. Biol. Chem. 256, 2143-2153) did not significantly affect the nonelectrostatic component but did perturb the electrostatic contribution to the binding energy (we are numbering the amino acid residues according to the DNA sequence). The former was determined by extrapolating the linear portion of the salt dependence curve (0.125 to 0.25 M KCl) to 1 M ionic strength, with the same result for both wild type and S187 endonucleases at both pH 6.0 and 7.4 (-8.5 +/- 1.5 kcal/mol or greater than 50% of the total binding free energy). The slopes of these same curves yield estimates of eight ionic interactions between wild type endonuclease and the DNA at both pH values. By contrast, binding of EcoRI-S187 to dodecanucleotide involves six charge-charge interactions at pH 6.0. Only two ionic interactions are observed at pH 7.4. This was unexpected since gel permeation chromatography demonstrated that the recognition complex for both wild type and S187 proteins contains an enzyme dimer and a DNA duplex. EcoRI-S187 endonuclease retains wild type DNA sequence specificity, and the rate of the phosphodiester hydrolysis step is also unchanged. Thus, electrostatic interactions are functionally separable from sequence recognition and strand cleavage. Our results also establish that arginine 187 plays a key role in the electrostatic function and suggest that it might be located at the DNA-protein interface. The disproportionate loss of ion pairs at pH 7.4 can be rationalized by a model which suggests that six conformationally mobile ionic groups on the protein act in a coordinated manner during the interaction with DNA.
Jacobson, L., and L. Jen-Jacobson (1982) Post-transcriptional control of protein synthesis during substrate adaptation. Pp 191-204 in Experiences in Biochemical Perception, Ornston, L.N., and S. Sligar, Ed. Academic Press, New York Jacobson, L.A., and L. Jen-Jacobson (1980) Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down. J. Bacteriol. 142:888-898 We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.
Jacobson, L.A., and L. Jen-Jacobson (1980) Control of protein synthesis in Escherichia coli: lack of correlation with changes in intracellular pools of ATP, GTP, and ppGpp. Arch. Biochem. Biophys. 203:691-696 |
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