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S Rajendran

Publications and source records attributed to S Rajendran.

At least 19 recordsLinked to original sources
PubMed2002

Insight into the development of non-adherent, absorbent dressings.

OBJECTIVE: This study aimed to develop a variety of wound dressing materials, made from standard natural fibres, that have high absorption and non-adherent characteristics. METHOD: A total of 21 dressings were made using knitted and crochet technologies and their absorbency was tested. Five non-adherent recipes were selected from a range of chemical formulations and the optimised non-adherent finishes were applied to the four best dressings. Their absorbency and non-adherent properties were evaluated. RESULTS: The study demonstrated that rib cotton (RC), rib viscose (RV), crochet cotton medium (CCM) and crochet viscose medium (CVM) dressings possess high absorption and that five finishing recipes, C + D, A + G, I, I + N and I + G, impart high absorption as well as non-adherent properties. The finish I + G is superior in imparting non-adherence to RV dressing, both in dry and moist conditions. This means that irrigation with water, saline or sodium citrate solution before removing the dressing from a wound is not needed. CONCLUSION: A number of novel knitted and crochet structures with enhanced absorbency have been designed for wound management using standard bleached fibres. Novel non-adherent finishes for the developed structures have been formulated for the developed dressings, and offer an alternative to existing non-adherent dressings.

Absorption↗
PubMed2001

Kinetic mechanisms of rat polymerase beta-ssDNA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (Pol beta)(16) and (Pol beta)(5) ssDNA binding mode.

Kinetics of rat polymerase beta (pol beta) binding to the single-stranded DNA (ssDNA) in the (pol beta)(16) and (pol beta)(5) binding modes has been examined, using the fluorescence stopped-flow technique. Binding of the enzyme to the ssDNA containing fluorescein is characterized by a strong increase of the DNA fluorescence, which provides an excellent signal to quantitatively study the complex mechanism of the ssDNA recognition process. The experiments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol beta)(16) binding mode, i.e. it encompasses the entire, total DNA-binding site of rat pol beta, and with a 10-mer which binds the enzyme exclusively in the (pol beta)(5) binding mode where only the 8 kDa domain of the enzyme is engaged in interactions with the DNA. The data indicate that the formation of the (pol beta)(16) binding mode occurs by a minimum three-step mechanism with the bimolecular binding step followed by two isomerizations: [formula-see text] A similar mechanism is observed in the formation of the (pol beta)(5) binding mode, although at low salt concentrations there is an additional, slow step in the reaction. The data analysis was performed using the matrix projection operator technique, a powerful method to address stopped-flow kinetics, particularly, amplitudes. The binding modes differ in the free energy changes of the partial reactions and ion effects on transitions between intermediates that reflect different participation of the two structural domains. The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol beta)(16) binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indicate that interactions of the ssDNA-binding subsite of the 8 kDa domain with the ssDNA, controlled by the ion binding, induce conformational transitions of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant conformational equilibrium of rat pol beta, prior to ssDNA binding.

Animals↗
PubMed2001

Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA binding modes.

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.

DNA Polymerase beta↗
PubMed2001

Recognition of template-primer and gapped DNA substrates by the human DNA polymerase beta.

Interactions between human DNA polymerase beta and the template-primer, as well as gapped DNA substrates, have been studied using quantitative fluorescence titration and analytical ultracentrifugation techniques. In solution, human pol beta binds template-primer DNA substrates with a stoichiometry much higher than predicted on the basis of the crystallographic structure of the polymerase-DNA complex. The obtained stoichiometries can be understood in the context of the polymerase affinity for the dsDNA and the two ssDNA binding modes, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotide residues occluded by the protein in the complex. The analysis of polymerase binding to different template-primer substrates has been performed using the statistical thermodynamic model which accounts for the existence of different ssDNA binding modes and has allowed us to extract intrinsic spectroscopic and binding parameters. The data reveal that the small 8 kDa domain of the enzyme can engage the dsDNA in interactions, downstream from the primer, in both (pol beta)(16) and (pol beta)(5) binding modes. The affinity, as well as the stoichiometry of human pol beta binding to the gapped DNAs is not affected by the decreasing size of the ssDNA gap, indicating that the enzyme recognizes the ssDNA gaps of different sizes with very similar efficiency. On the basis of the obtained results we propose a plausible model for the gapped DNA recognition by human pol beta. The enzyme binds the ss/dsDNA junction of the gap, using its 31 kDa domain, with slight preference over the dsDNA. Binding only to the junction, but not to the dsDNA, induces an allosteric conformational transition of the enzyme and the entire enzyme-DNA complex which results in binding of the 8 kDa domain with the dsDNA. This, in turn, leads to the significant amplification of the enzyme affinity for the gap over the surrounding dsDNA, independent of the gap size. The presence of the 5'-terminal phosphate, downstream from the primer, has little effect on the affinity, but profoundly affects the ssDNA conformation in the complex. The significance of these results for the mechanistic model of the functioning of human pol beta is discussed.

Allosteric Regulation↗
PubMed2001

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.

Animals↗
PubMed2001

Energetics and specificity of Rat DNA polymerase beta interactions with template-primer and gapped DNA substrates.

Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.

Animals↗
PubMed2001

The action of phosphine against the eggs of phosphine-resistant and -susceptible strains of Rhyzopertha dominica F.

The effect of phosphine at exposure periods of 24, 48 and 120 h on hatching and mortality of 0- to 1-day-old eggs of susceptible (TN6) and resistant (FC10) strains of Rhyzopertha dominica of field origin was investigated. The fumigant affected hatching in both the strains. In a 48-h exposure at 27 (+/- 2) degrees C, the LD99 doses for the eggs of TN6 and FC10 were 0.56 and 3.25 mg litre-1, respectively. Significant reduction in hatching was observed in treated batches with progressive increase of phosphine dose in the first 2-3 days. On subsequent days the numbers hatching were often similar to those in controls, and sometimes exceeded control hatch, especially following a 5-day exposure. A critical change in the order of susceptibility of egg and adult stages of the two strains was noticed. In 48-h exposures, eggs of the susceptible strain were more tolerant than their adults, whilst the reverse was true in the resistant strain.

Animals↗
PubMed2001

Performance of phosphine in fumigation of bagged paddy rice in indoor and outdoor stores.

Phosphine fumigation trials were carried out on bag-stacks of paddy rice to study the differences in gas loss rates and concentration-time (Ct) products achieved during the treatment of indoor and outdoor stacks. Stacks (89-132t) were fumigated singly under 250&mgr;m thick polyethylene sheeting, which was sealed with a double layer of sand-snakes to the concrete floor. Phosphine was applied as an aluminium phosphide formulation and the fumigations continued for 7 days. In the first experiment, stacks of paddy rice with moisture contents ranging from 12.2 to 13.7% were held in either indoor or in outdoor storage and subjected to fumigation at the rate of 2, 3 or 4g of phosphine/tonne. The outdoor stacks held relatively low levels of phosphine with Ct products for the indoor stacks of 135, 171 and 294gh/m(3), respectively, whilst the corresponding values for the outdoor stacks were 70, 85 and 166gh/m(3) only. The average gas loss rate was 14.5% per day for the indoor stacks and 29.5% for the outdoor stacks. In the second experiment, old stacks of paddy rice inside a godown, one each with grains at 8.8 and 9.8% moisture content, were fumigated at 3g phosphine/tonne. Release of phosphine was delayed and fumigant sorption was less and therefore higher Ct products of 204 and 216gh/m(3) were achieved. In the stacks built outdoors, the resident infestations of Rhyzopertha dominica, Cryptolestes sp. and Oryzaephilus surinamensis were completely controlled despite lower Ct products. On the other hand, in the stacks of old paddy, R. dominica survived the treatment. Subsequent testing showed that the population had a degree of resistance to phosphine.

Journal Article↗
PubMed2000

Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: quantitative analysis using the rapid quench-flow method.

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined by:¿eqalign¿¿¿rm Helicase+ATP¿&¿mathop¿¿rightleftharpoons¿ ¿k_1¿_¿k_¿-1¿¿¿¿rm (H-ATP)¿¿mathop¿¿rightleftharpoons¿ ¿k_2¿_¿k_¿-2¿¿¿¿rm (H-ADP¿cdot Pi)¿¿cr &¿mathop¿¿rightleftharpoons¿ ¿k_3¿_¿k_¿-3¿¿¿¿rm (H-ADP¿cdot Pi)¿ *¿The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP)=(1.3+/-0.5)x10(5) M(-1). The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H)=k(2)/k(-2) approximately 2 while the subsequent conformational transition is characterized by K(3)=k(3)/k(-3) approximately 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release.The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter sigma=0.25(+/-0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed.

Adenosine Triphosphatases↗
PubMed2000

Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein.

Quantitative analyses of the interactions of nucleotide cofactors with the Escherichia coli replicative factor DnaC protein have been performed using thermodynamically rigorous fluorescence titration techniques. This approach allowed us to obtain stoichiometries of the formed complexes and interaction parameters, without any assumptions about the relationship between the observed signal and the degree of binding. The stoichiometry of the DnaC-nucleotide complex has been determined in direct binding experiments with fluorescent nucleotide analogues, MANT-ATP and MANT-ADP. The stoichiometry of the DnaC complexes with unmodified ATP and ADP has been determined using the macromolecular competition titration method (MCT). The obtained results established that at saturation the DnaC protein binds a single nucleotide molecule per protein monomer. Analyses of the binding of fluorescent analogues and unmodified nucleotides to the DnaC protein show that ATP and ADP have the same affinities for the nucleotide-binding site, albeit the corresponding complexes have different structures, specifically affected by the presence of magnesium cations in solution. Although the presence of the gamma-phosphate does not affect the affinity, the structure of the triphosphate group is critical. While the affinity of ATP-gamma-S is the same as the affinity of ATP, the affinities of AMP-PNP and AMP-PCP are approximately 2 and approximately 4 orders lower than that of ATP, respectively. Moreover, the ribose plays a significant role in forming a stable complex. The binding constants of dATP and dADP are approximately 2 orders of magnitude lower than those for ribose nucleotides. The nucleotide-binding site of the DnaC protein is highly base specific. The intrinsic affinity of adenosine triphosphates and diphosphates is at least 3-4 orders of magnitude higher than for any of the other examined nucleotides. The obtained data indicate that the recognition mechanism of the nucleotide by the structural elements of the binding site is complex with the base providing the specificity and the ribose, as well as the second phosphate group contributing to the affinity. The significance of the results for the functioning of the DnaC protein is discussed.

Adenosine↗
PubMed2000

Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities.

Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.

Binding Sites↗
PubMed2000

A survey of some Indian medicinal plants for anti-human immunodeficiency virus (HIV) activity.

BACKGROUND & OBJECTIVES: India has an extensive area of forest enriched with plant diversity. Several of these plants have been used as folklore medicines. However, the medicinal plants have rarely been investigated for anti-human immunodeficiency virus activity. Hence, some Indian medicinal plants were screened in vitro against human immunodeficiency virus (HIV). METHODS: The inhibitory effect of plant extracts on HIV replication was monitored in terms of inhibition of virus induced cytopathogenicity in MT-4 cells. The MT-4 cells were infected with HIV. The HIV infected or mock infected MT-4 cells were incubated at 37 degrees C in a CO2 incubator in the presence of the plant extracts. After five days, cell viability was measured by tetrazolium based colorimetric assay. RESULTS & INTERPRETATION: Of the 69 plant species screened, 16 were effective against HIV-1 and 4 were against both HIV-1 and HIV-2. The most effective extracts against HIV-1 and HIV-2 are respectively Cinnamomum cassia (bark) and Cardiospermum helicacabum (shoot + fruit). The findings provide a rationale for further studies on isolation of active principles and pharmacological evaluation.

Anti-HIV Agents↗
PubMed1999

Familial Lecithin:cholesterol acyltransferase deficiency with renal failure in two siblings. First case report from India.

We encountered 2 siblings with corneal opacities, anemia, decreased concentration of plasma esterified cholesterol, chronic renal failure and markedly reduced levels of plasma lecithin:cholesterol acyltransferase. Renal biopsy in 1 patient showed clear lacunae containing characteristic dense bodies in the glomerular and tubular basement membranes and the interstitium. One of the 2 siblings received a kidney transplant and is doing well after 9 years. This is the first report of this rare metabolic disease from India.

Adult↗
PubMed1999

Endodermal sinus tumour of the ovary in pregnancy.

We present a case of a 30-year-old pregnant woman in whom an ovarian mass was identified by ultrasonography at 15 weeks' gestation. A markedly elevated maternal serum alphafetoprotein (MSAFP) suggested a diagnosis of endodermal sinus tumour of the ovary. A right salpingo-oophorectomy at 19 weeks' gestation enabled histological confirmation of the diagnosis and suggested a stage 1 tumour. Unfortunately tumour recurrence necessitated further laparotomy and delivery by caesarean section at 32 weeks' gestation. A total abdominal hysterectomy and left salpingo-oophorectomy was undertaken with resection of the splenic flexure and formation of a double-barrelled colostomy after which no gross intraperitoneal tumour remained. Three weeks later a new suprahepatic tumour mass was excised and the colostomy was closed. The patient then received four cycles of combination chemotherapy with cisplatin, etoposide, and bleomycin. Unfortunately she developed a faecal fistula at the site of the colostomy. Germ cell tumours are highly responsive to chemotherapy. Over-zealous surgery particularly involving bowel resection may cause unnecessary morbidity and compromise the outcome.

Adult↗
PubMed1998

Transition between different binding modes in rat DNA polymerase beta-ssDNA complexes.

Interactions of rat DNA polymerase beta with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol beta-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol beta forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol beta binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol beta)16 binding mode and the second as the (pol beta)5 binding mode. The formation of the (pol beta)16 and (pol beta)5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol beta)16 or the (pol beta)5 mode, and a 10mer which can form only the (pol beta)5 mode. Binding of rat pol beta to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol beta)5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol beta)5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol beta)16 than the (pol beta)5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol beta)16 mode. The significance of these results for understanding the functioning of rat pol beta in the DNA metabolism is discussed.

Animals↗
PubMed1998

Human DNA polymerase beta recognizes single-stranded DNA using two different binding modes.

Interactions between the human DNA polymerase beta (pol beta) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(depsilonA)) as a function of the binding density of pol beta-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol beta forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol beta binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol beta)16 and the second as (pol beta)5 binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol beta binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol beta, is discussed.

Allosteric Regulation↗
PubMed1998

Does single-stranded DNA pass through the inner channel of the protein hexamer in the complex with the Escherichia coli DnaB Helicase? Fluorescence energy transfer studies.

The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5' arm of the replication fork, without ATP hydrolysis, the distance between the 5' end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5' arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2-3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.

Bacterial Proteins↗