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T M Lohman

Publications and source records attributed to T M Lohman.

At least 55 records · Page 3Linked to original sources
PubMed1993

Heterodimer formation between Escherichia coli Rep and UvrD proteins.

DNA helicases catalyze the essential process of unwinding duplex DNA to form the single-stranded DNA intermediates required for DNA metabolic processes including replication, recombination, and repair. Most cells, possibly all, encode multiple helicases that function selectively in different processes, although some helicases can complement each other in vivo. Thus, although Escherichia coli can survive mutations or deletions of either the uvrD gene (encoding Helicase II) or the rep gene (encoding Rep helicase) separately, deletion of both rep and uvrD genes is lethal (Washburn, B. K., and Kushner, S. R. (1991) J. Bacteriol. 173,2569-2575). The Rep and UvrD polypeptides share approximately 40% sequence homology, and we have previously shown that both form homodimeric species and that the Rep homodimer appears to be the functionally active helicase. We report here that these two proteins can also interact in vitro to form a heterodimer. The heterodimer appears to be energetically more stable than the Rep homodimer but less stable than the UvrD homodimer under our conditions. The observation of Rep/UvrD heterodimer formation in vitro opens up the intriguing possibility that the heterodimer may play a physiologically important role that is distinct from the role of the Rep or UvrD homodimers. Therefore, considerations of the role of either protein in DNA metabolic processes, such as replication and repair, must include a potential role for a Rep/UvrD heterodimer.

Adenosine Triphosphatases↗
PubMed1993

Escherichia coli rep helicase unwinds DNA by an active mechanism.

DNA helicases unwind duplex DNA to form the single-stranded (ss) DNA intermediates required for replication, recombination, and repair in reactions that require nucleoside 5'-triphosphate hydrolysis. Helicases generally require a ss-DNA flanking the duplex in order to initiate unwinding in vitro; however, the precise function of the ss-DNA is not understood. If a helicase unwinds DNA by a "passive" mechanism, it would bind to and translocate unidirectionally along the ss-DNA and facilitate duplex unwinding by translocating onto the ss-DNA that is formed transiently by thermal fluctuations in the duplex. We have examined the kinetics of DNA unwinding by Escherichia coli Rep protein (a 3' to 5' helicase) by rapid quench-flow methods using a series of novel, nonnatural DNA substrates possessing 3' flanking ss-DNA within which is embedded either a segment of ss-DNA possessing reversed backbone polarity or a non-DNA [poly(ethylene glycol)] spacer, either of which should block unwinding by a passive helicase. The E. coli Rep helicase effectively unwinds these DNA substrates, ruling out a passive mechanism of unwinding. Instead, the results are consistent with an "active" rolling mechanism during which Rep binds to ss-DNA and duplex DNA simultaneously.

Adenosine Triphosphatases↗
PubMed1993

A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions.

Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision. To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane. Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point. The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology. The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered. This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered. The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered. The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides.

Base Sequence↗
PubMed1993

Kinetics of Escherichia coli helicase II-catalyzed unwinding of fully duplex and nicked circular DNA.

Escherichia coli helicase II (UvrD) protein can initiate unwinding of duplex DNA at blunt ends or nicks, although these reactions require excess protein. We have undertaken kinetic studies of these reactions in order to probe the mechanism of initiation of unwinding. DNA unwinding was monitored directly by using agarose gel electrophoresis and indirectly through the rate of ATP hydrolysis by helicase II in the presence of an ATP-regenerating system. In the presence of fully duplex DNA and excess helicase II, the rate of ATP hydrolysis displays a distinct lag phase before the final steady-state rate of hydrolysis is reached. This reflects the fact that ATP hydrolysis under these conditions results from helicase II binding to the ssDNA products of the unwinding reaction, rather than from an intrinsic duplex DNA-dependent ATPase activity. Unwinding of short blunt-ended duplex DNA (341 and 849 base pairs) occurs in an "all-or-none" reaction, indicating that initiation of unwinding by helicase II is rate-limiting. We propose a minimal mechanism for the initiation of DNA unwinding by helicase II which includes a binding step followed by the rate-limiting formation of an initiation complex, possibly involving protein dimerization, and we have determined the phenomenological kinetic parameters describing this mechanism. Unwinding of a series of DNA substrates containing different initiation sites (e.g., blunt ends, internal nicks, and four-nucleotide 3' vs 5' ssDNA flanking regions) indicates that the rate of initiation is slowest at nicks and, surprisingly, at ends possessing a four-nucleotide 3' ssDNA flanking region.

Adenosine Triphosphatases↗
PubMed1993

Helicase-catalyzed DNA unwinding.

DNA helicases are ubiquitous and multiple helicases have been identified in a number of prokaryotes and eukaryotes. Although it is clear that not all helicases function identically, many of these enzymes possess similar properties that appear to be of general importance for their mechanism of action. For example, the assembly states of most (possibly all) helicases are oligomeric. The prime consequence of an oligomeric helicase is that it possesses multiple DNA binding sites, a feature that is required for any "active" mechanism of DNA unwinding, since it enables a helicase to bind both ss- and duplex DNA or two strands of ss-DNA simultaneously at an unwinding fork. Modulation of the relative affinities of ss- versus duplex DNA for these multiple binding sites through ATP binding and hydrolysis, as has been observed for the E. coli Rep dimer, can provide a mechanism for translocation and processive unwinding of DNA. Along with studies of DNA unwinding, further understanding of helicase mechanisms requires quantitative studies of the equilibria and kinetics of the multiple, linked reactions among protein, DNA, and nucleotide cofactors, including the protein-protein interactions involved in assembly of the oligomeric helicase.

Adenosine Triphosphatases↗
PubMed1993

Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II).

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.

Adenosine Triphosphatases↗
PubMed1992

Thermodynamics of single-stranded RNA binding to oligolysines containing tryptophan.

The equilibrium binding to the synthetic RNA poly(U) of a series of oligolysines containing one, two, or three tryptophans has been examined as a function of pH, monovalent salt concentration (MX), temperature, and Mg2+. Oligopeptides containing lysine (K) and tryptophan (W) of the type KWKp-NH2 and KWKp-CO2 (p = 1-8), as well as peptides containing additional tryptophans or glycines, were studied by monitoring the quenching of the peptide tryptophan fluorescence upon binding poly(U). Equilibrium association constants, K(obs), and the thermodynamic quantities delta G(o)obs, delta H(o)obs, and delta S(o)obs describing peptide-poly(U) binding were measured as well as their dependences on monovalent salt concentration, temperature, and pH. In all cases, K(obs) decreases significantly with increasing monovalent salt concentration, with (delta log K(obs)/delta log [K+]) = -0.74 (+/- 0.04)z, independent of temperature and salt concentration, where z is the net positive charge on the peptide. The origin of these salt effects is entropic, consistent with the release of counterions from the poly(U) upon formation of the complex. Upon extrapolation to 1 M K+, the value of delta G(o)obs is observed to be near zero for all oligolysines binding to poly(U), supporting the conclusion that these complexes are stabilized at lower salt concentrations due to the increase in entropy accompanying the release of monovalent counterions from the poly(U). Only the net peptide charge appears to influence the thermodynamics of these interactions, since no effects of peptide charge distribution were observed. The binding of poly(U) to the monotryptophan peptides displays interesting behavior as a function of the peptide charge. The extent of tryptophan fluorescence quenching, Qmax, is dependent upon the peptide charge for z less than or equal to +4, and the value of Qmax correlates with z-dependent changes in delta H(o)obs and delta S(o)obs(1 M K+), whereas for z greater than or equal to +4, Qmax, delta H(o)obs, and delta S(o)obs (1 M K+) are constant. The correlation between Qmax and delta H(o)obs and delta S(o)obs(1 M K+) suggests a context (peptide charge)-dependence of the interaction of the peptide tryptophan with poly(U). However the interaction of the peptide tryptophan does not contribute substantially to delta G(o)obs for any of the peptides, independent of z, due to enthalpy-entropy compensations. Each of the tryptophans in multiple Trp-containing peptides appear to bind to poly(U) independently, with delta H(o)Trp = -2.9 +/- 0.7, although delta G(o)Trp is near zero due to enthalpy-entropy compensations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cations, Monovalent↗
PubMed1992

Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions.

The Escherichia coli single-strand binding (SSB) protein is an essential protein involved in DNA replication, recombination, and repair processes. The tetrameric protein binds to ss nucleic acids in a number of different binding modes in vitro. These modes differ in the number of nucleotides occluded per SSB tetramer and in the type and degree of cooperative complexes that are formed with ss DNA. Although it is not yet known whether only one or all of these modes function in vivo, based on the dramatically different properties of the SSB tetramer in these different ss DNA binding modes, it has been suggested that the different modes may function selectively in replication, recombination, and/or repair. The transitions between these different modes are very sensitive to solution conditions, including salt (concentration, as well as cation and anion type), pH, and temperature. We have examined the effects of multivalent cations, principally the polyamine spermine, on the SSB-ss poly(dT) binding mode transitions and find that the transition from the (SSB)35 to the (SSB)56 binding mode can be induced by micromolar concentrations of polyamines as well as the inorganic cation Co(NH3)6(3+). Furthermore, these multivalent cations, as well as Mg2+, induce the binding mode transition by binding cooperatively to the SSB-poly(dT) complexes. These observations are interesting in light of the fact that polyamines, such as spermidine, are part of the ionic environment in E. coli and hence these cations are likely to affect the distribution of SSB-ss DNA binding modes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗
PubMed1992

Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding.

The Escherichia coli Rep helicase unwinds duplex DNA during replication. The functional helicase appears to be a dimer that forms only on binding DNA. Both protomers of the dimer can bind either single-stranded or duplex DNA. Because binding and hydrolysis of adenosine triphosphate (ATP) are essential for helicase function, the energetics of DNA binding and DNA-induced Rep dimerization were studied quantitatively in the presence of the nucleotide cofactors adenosine diphosphate (ADP) and the nonhydrolyzable ATP analog AMPP(NH)P. Large allosteric effects of nucleotide cofactors on DNA binding to Rep were observed. Binding of ADP favored Rep dimers in which both protomers bound single-stranded DNA, whereas binding of AMPP(NH)P favored simultaneous binding of both single-stranded and duplex DNA to the Rep dimer. A rolling model for the active unwinding of duplex DNA by the dimeric Rep helicase is proposed that explains vectorial unwinding and predicts that helicase translocation along DNA is coupled to ATP binding, whereas ATP hydrolysis drives unwinding of multiple DNA base pairs for each catalytic event.

Adenine Nucleotides↗
PubMed1992

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1992

Thermodynamics of ligand-nucleic acid interactions.

Ligand-and protein-DNA equilibria are extremely sensitive to solution conditions (e.g., salt, temperature, and pH), and, in general, the effects of different solution variables are interdependent (i.e., linked). As a result, an assessment of the basis for the stability and specificity of ligand-or protein-DNA interactions requires quantitative studies of these interactions as a function of a range of solution variables. Many of the most dramatic effects on the stability of these interactions result from changes in the entropy of the system, caused by the preferential interaction of small molecules, principally ions which are released into solution on complex formation. A determination of the contributions of these entropy changes to the stability and specificity of protein-and ligand-DNA interactions requires thermodynamic approaches and cannot be assessed from structural studies alone.

Binding Sites↗
PubMed1992

Escherichia coli DNA helicases: mechanisms of DNA unwinding.

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.

Binding Sites↗
PubMed1991

DNA-induced dimerization of the Escherichia coli Rep helicase.

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 microM (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-gamma-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 less than or equal to n less than or equal to 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n less than or equal to 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.

Adenosine Triphosphatases↗
PubMed1991

Monomer-tetramer equilibrium of the Escherichia coli ssb-1 mutant single strand binding protein.

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/2 = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with delta H0 = -51 +/- 7 kcal/mol (pH 8.1) and delta H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative delta H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

DNA-Binding Proteins↗
PubMed1991

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA.

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

Bacterial Proteins↗
PubMed1991

Thermodynamic methods for model-independent determination of equilibrium binding isotherms for protein-DNA interactions: spectroscopic approaches to monitor binding.

The measurement of equilibrium binding constants for ligand-macromolecule interactions by monitoring a change in some spectral property of the ligand or the macromolecule is a common method used to study these interactions. This is due to the high sensitivity of the spectroscopic methods and general ease in applying these experimental procedures. In addition, binding can be monitored continuously, thus facilitating kinetic measurements. The main problem with these methods results from the fact that the spectroscopic signal is an indirect measure of binding, since the relationship between the change in the spectroscopic signal and the extent of binding is unknown, a priori. A common recourse is to assume a strict proportionality between the signal change and the fractional saturation of the ligand or macromolecule; however, it is often the case that such a direct proportionality does not hold. In this chapter we have reviewed the use of methods to analyze ligand-macromolecule equilibrium titrations that are monitored by indirect spectroscopic techniques. These methods of analysis yield thermodynamically rigorous, model-independent binding isotherms, hence assumptions concerning the relationship between the signal change and the extent of binding are not required. In fact, these methods can also be used to determine quantitatively the relationship between the signal change and the average degree of binding. In addition, the approaches discussed here are general and not limited to spectroscopic signals and therefore can be used with any intensive physicochemical property that reflects binding.

DNA↗
PubMed1990

DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins.

The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin. Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S). The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities. When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities. This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA. This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA. The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides. Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N. & Kornberg, A. (1981) J. Biol. Chem. 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities. However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein. Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions. This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein. The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin. Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗