Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.     

Selective binding of amino acid residues to tRNAPhe

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.     

Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clustering of physical health multimorbidity in people with severe mental illness: An accumulated prevalence analysis of United Kingdom primary care data

BackgroundPeople with severe mental illness (SMI) have higher rates of a range of physical health conditions, yet little is known regarding the clustering of physical health conditions in this population. We aimed to investigate the prevalence and clustering of chronic physical health conditions in people with SMI, compared to people without SMI.Methods and findingsWe performed a cohort-nested accumulated prevalence study, using primary care data from the Clinical Practice Research Datalink (CPRD), which holds details of 39 million patients in the United Kingdom. We identified 68,783 adults with a primary care diagnosis of SMI (schizophrenia, bipolar disorder, or other psychoses) from 2000 to 2018, matched up to 1:4 to 274,684 patients without an SMI diagnosis, on age, sex, primary care practice, and year of registration at the practice. Patients had a median of 28.85 (IQR: 19.10 to 41.37) years of primary care observations. Patients with SMI had higher prevalence of smoking (27.65% versus 46.08%), obesity (24.91% versus 38.09%), alcohol misuse (3.66% versus 13.47%), and drug misuse (2.08% versus 12.84%) than comparators. We defined 24 physical health conditions derived from the Elixhauser and Charlson comorbidity indices and used logistic regression to investigate individual conditions and multimorbidity. We controlled for age, sex, region, and ethnicity and then additionally for health risk factors: smoking status, alcohol misuse, drug misuse, and body mass index (BMI). We defined multimorbidity clusters using multiple correspondence analysis (MCA) and K-means cluster analysis and described them based on the observed/expected ratio. Patients with SMI had higher odds of 19 of 24 conditions and a higher prevalence of multimorbidity (odds ratio (OR): 1.84; 95% confidence interval [CI]: 1.80 to 1.88, p < 0.001) compared to those without SMI, particularly in younger age groups (males aged 30 to 39: OR: 2.49; 95% CI: 2.27 to 2.73; p < 0.001; females aged 18 to 30: OR: 2.69; 95% CI: 2.36 to 3.07; p < 0.001). Adjusting for health risk factors reduced the OR of all conditions. We identified 7 multimorbidity clusters in those with SMI and 7 in those without SMI. A total of 4 clusters were common to those with and without SMI; while 1, heart disease, appeared as one cluster in those with SMI and 3 distinct clusters in comparators; and 2 small clusters were unique to the SMI cohort. Limitations to this study include missing data, which may have led to residual confounding, and an inability to investigate the temporal associations between SMI and physical health conditions.ConclusionsIn this study, we observed that physical health conditions cluster similarly in people with and without SMI, although patients with SMI had higher burden of multimorbidity, particularly in younger age groups. While interventions aimed at the general population may also be appropriate for those with SMI, there is a need for interventions aimed at better management of younger-age multimorbidity, and preventative measures focusing on diseases of younger age, and reduction of health risk factors.

In an observational analysis of primary care data from the UK, Naomi Launders and colleagues study the prevalence and clustering of physical health conditions and multimorbidity in individuals with severe mental illnesses.     

On the cooperative binding of large ligands to a one-dimensional homogeneous lattice: the generalized three-state lattice model

We present the general secular equation for three-state lattice models for the cooperative binding of large ligands to a one-dimensional lattice. In addition, a closed-form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter omega(0 less than omega less than infinity) thus eliminating the need for multiple equations.     

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.     

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects

We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant KN, and the negative co-operativity constant, sigma N. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. delta log KN/delta log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that delta Ho = -26(+/- 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. sigma N less than 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of sigma 16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for sigma 16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.     

Interactions of the Escherichia coli DnaB helicase hexamer with the replication factor the DnaC protein. Effect of nucleotide cofactors and the ssDNA on protein-protein interactions and the topology of the complex

Quantitative studies of interactions between the Escherichia coli replication factor DnaC protein and the DnaB helicase have been performed using sedimentation velocity and fluorescence energy transfer techniques. The applied novel analysis of the sedimentation data allows us to construct thermodynamic rigorous binding isotherms without any assumption as to the relationship between the observed molecular property of the complexes formed, the average sedimentation coefficient, or the degree of binding. Experiments have been performed with the fluorescein-modified DnaB helicase, which allows an exclusive monitoring of the DnaB-DnaC complex formation. The DnaC binding to the unmodified helicase has been characterized in competition experiments. The data establish that, in the presence of the ATP analog AMP-PNP, or ADP, a maximum of six DnaC monomers bind cooperatively to the DnaB hexamer. The positive cooperative interactions are limited to the two neighboring DnaC molecules. Analyses using a statistical thermodynamic hexagon model indicate that, under the solution conditions examined, the affinity is characterized by the intrinsic binding constant K=1.4(+/-0.5)x10(5)M(-1) and cooperativity parameter sigma=21+/-5. These data suggest strongly that the DnaC-DnaB complex exists in vivo as a mixture of complexes with a different number of bound DnaC molecules, although the complex with six DnaC molecules bound dominates the distribution. The DnaC nucleotide-binding site is not involved in the stabilization of the complex. Moreover, the hydrolysis of NTP bound to the helicase or the DnaC is not required for the release of the DnaC protein from the complex. The single-stranded DNA (ssDNA) bound to the helicase does not affect the DnaC protein binding. However, in the presence of the DNA, there is a significant difference in the energetics and structure of the ternary complex, DnaC-DnaB-ssDNA, formed in the presence of AMP-PNP as compared to ADP. The topology of the ternary complex DnaC-DnaB-ssDNA has been determined using the fluorescence energy transfer method. In solution, the DnaC protein-binding site is located on the large 33 kDa domain of the DnaB helicase. The significance of the results in the functioning of the DnaB helicase-DnaC protein complex is discussed.     

Multistep sequential mechanism of Escherichia coli helicase PriA protein-ssDNA interactions. Kinetics and energetics of the active ssDNA-searching site of the enzyme

Kinetics of the Escherichia coli PriA helicase interactions with the ssDNA has been studied, using the fluorescence stopped-flow technique. Experiments have been performed with a series of fluorescent etheno derivatives of ssDNA adenosine oligomers, differing in the number of nucleotide residues. The PriA helicase binds the ssDNA in the sequential process defined by [reaction: see text]. In the first step, the enzyme associates fast with the ssDNA without inducing conformational changes in the DNA. The dependence of the partial equilibrium constant, characterizing the first step, upon the length of the ssDNA strictly reflects the statistical relationship between the size of the DNA-binding site and the number of potential binding sites on the ssDNA. Only the DNA-binding site that encompasses 6.3 +/- 1 residues is directly involved in interactions. The site is located on a structural domain allowing the enzyme to efficiently search and recognize small patches of the ssDNA. Intramolecular steps are independent of the ssDNA length and accompanied by changes in the DNA structure. Salt and glycerol effects on the studied kinetics indicate a very different nature of the intermediates. While the bimolecular step is characterized by net ion release and water uptake, net ion uptake and water release accompany intramolecular transitions. Specific ion binding stabilizes the helicase-ssDNA complex in (P)(2) and (P)(3) intermediates. However, magnesium and AMP-PNP do not affect the mechanism of enzyme-ssDNA interactions. The sequential character of the mechanism indicates that the enzyme does not exist in a preequilibrium conformational transition prior to the DNA binding.     

UNC-45B Chaperone: The Role of its Domains in the Interaction with the Myosin Motor Domain

The proper folding of many proteins can only be achieved by interaction with molecular chaperones. The molecular chaperone UNC-45B is required for the folding of striated muscle myosin II. However, the precise mechanism by which it contributes to proper folding of the myosin head remains unclear. UNC-45B contains three domains: an N-terminal TPR domain known to bind Hsp90, a Central domain of unknown function, and a C-terminal UCS domain known to interact with the myosin head. Here we used fluorescence titrations methods, dynamic light scattering, and single-molecule atomic force microscopy (AFM) unfolding/refolding techniques to study the interactions of the UCS and Central domains with the myosin motor domain. We found that both the UCS and the Central domains bind to the myosin motor domain. Our data show that the domains bind to distinct subsites on the myosin head, suggesting distinct roles in forming the myosin−UNC-45B complex. To determine the chaperone activity of the UCS and Central domains, we used two different methods: 1), prevention of misfolding using single-molecule AFM, and 2), prevention of aggregation using dynamic light scattering. Using the first method, we found that the UCS domain is sufficient to prevent misfolding of a titin mechanical reporter. Application of the second method showed that the UCS domain but not the Central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS and the Central domains bind the myosin head with high affinity, only the UCS domain displays chaperone activity.