Echocardiographic Assessment of Valvular Involvement in Chronic Rheumatic Heart Disease in BSMMU, Bangladesh.

Rheumatic heart disease (RHD) is a disease of poverty, is almost entirely preventable, and is the most common cardiovascular disease worldwide in those under 25 years especially in the developing county like Bangladesh. RHD is caused by acute rheumatic fever (ARF) which typically results in cumulative valvular lesions that may present clinically after a number of years of sub-clinical disease. It has a progressive course and patients usually may require valve repair/replacement in future. Echocardiography is an easily available, non-invasive, widely used, standard tool for diagnosis and evaluation of RHD. But there is scarcity of echocardiographic study of Valvular Involvement in Chronic Rheumatic Heart Disease (CHRD) in Bangladesh. This study was aimed to utilize echocardiography as a tool to evaluate patients of CRHD in Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. This observational study was conducted in the Department of Cardiology, BSMMU from September 2018 to August 2019. Echocardiography was done in each patient only once with VividE9®machine. Among 1350 echocardiography, 101 patients (7.5%) were diagnosed as RHD including post valve replacement patients. The mean age of the patients was 40±14 years and 64.34% were female. Mitral stenosis (MS) was the commonest lesion in 84.15% followed by mitral regurgitation (MR) in 66.33%, tricuspid regurgitation (TR) in 57.43%, aortic regurgitation (AR) in 49.51%, aortic stenosis (AS) in 26.74% and pulmonary regurgitation in 10.89%. The frequency of complications like pulmonary hypertension, heart failure, atrial fibrillation (AF), LA thrombus, stroke and infective endocarditis was 67.33%, 61.05%, 18.81%, 6.93%, 3.96% and 0.99% respectively. History of Rheumatic fever was present only in 10.89% patient. Mitral stenosis was the commonest lesion seen mostly in female and most common complication was pulmonary hypertension. Mean age of patients in this study was higher than other contemporary studies and frequency as well as severity of complications was also more in female.

Feasibility and Safety of Distal Transradial Access in the Anatomical Snuffbox for Coronary Angiography and Intervention.

Distal transradial access in the anatomical snuffbox has advantages over standard proximal access in terms of patient and operator comfort levels and risk of ischemia. Radial artery preservation could be a relevant issue in patients requiring multiple radial artery procedures and coronary bypass with the use of a radial graft or construction of Arterio-Venous fistula in patient of chronic kidney disease. One relevant drawback is the challenging puncture of a small and weak artery, with a steeper learning curve. The study was aimed at proving feasibility and safety of distal transradial access in the anatomical snuffbox. A total of 100 patients were assigned to perform coronary angiogram or intervention through distal transradial access in the anatomical snuffbox from January 2018 to June 2018 in this unit of the University Cardiac Center (UCC), Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. All of them had normal pulse in their distal radial artery. Both right and left distal radial artery were used and demographic features & complications were recorded during hospital stay. Statistical analysis was done through SPSS version 19. The overall feasibility was 98%, greater than expected in this early clinical experience, with 98 successful accesses out of 100 patients. There was failure to access of distal radial artery in two cases which may be due to hypoplastic/vasospastic distal radial artery. Despite all it can be said that it was very much safe as there was no hand ischemia, hematoma, numbness or proximal radial arterial occlusion. Distal transradial access in the anatomical snuffbox for coronary angiography and intervention is a safe and feasible option for both patients and operators.

Visceral Adiposity Index Score is the Better Predictor of Clinical and Coronary Angiographic Severity Assessment than Other Adiposity Indices in Patients with Acute Coronary Syndrome.

Acute coronary syndrome (ACS) is one of the leading causes of death throughout the world and obesity especially visceral adiposity is one of the important concerns globally due to its huge impact on coronary artery disease particularly on ACS. There are several traditional methods like BMI, WC, WHR, WHtR etc. but none of these can measure visceral fat accurately. In this regard visceral adiposity index (VAI) is a novel sex specific index which has significant correlation with visceral adiposity and can express the distribution as well as function of visceral fat precisely. This cross sectional study was done in the Cardiology Department of National Institute of Cardiovascular Diseases, Dhaka, Bangladesh from August 2015 to July 2016 to compare the VAI with other adiposity indices for clinical and coronary angiographic severity assessment in patients with acute coronary syndrome. A total of 200 patients (Case 100 patients of ACS and Control 100 patients of non ACS) were included. Afterward clinical, biochemical, echocardiographic and coronary artery angiographic indexes determined by Gensini score were acquired. Adiposity indices like BMI, Waist and Hip circumference, Waist Hip and Waist Height ratio (WHR, WHtR) and finally VAI were calculated using appropriate formula. Patient with ACS had more severe form of clinical features like severe chest pain & shortness of breath (p=0.001), pulse, BP, abnormal precordial findings, BMI, WC, WHR, WHtR, HC, VAI (p=0.001) and angiographic severity (p=0.001) than non ACS group. Multivariate binary logistic regression analysis for clinical and coronary angiographic severity assessment (GS>36) by adiposity indices showed VAI was the better predictor of clinical and coronary angiographic severity assessment with OR's being 5.61 than others. An ROC curve was plotted for each adiposity indices for clinical and coronary angiographic severity assessment showed VAI to have the maximal AUC. A VAI of OR-5.61 was provided as the cutoff value which had a sensitivity of 73.3% and specificity of 76.6% (AUROC=0.839, CI-0.760-0.918, p<0.001) which indicates better than other adiposity indices in patients under study. VAI is an excellent, simple, noninvasive tool to detect the visceral adipose mass & was markedly associated with the clinical and coronary angiographic severity assessment in patients with ACS.

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides. The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity. In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other. Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS. DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1. Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs. Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.

A novel human hexameric DNA helicase: expression, purification and characterization.

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >/=5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5'-->3' direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.

Can infants and young children eat enough green leafy vegetables from a single traditional meal to meet their daily vitamin A requirements?

To evaluate the feasibility of providing adequate vitamin A precursors to meet the daily need from a meal oftraditionally cooked green leafy vegetables and boiled rice and to understand mothers' perceptions and acceptance of leafy vegetables for infants and young children, 118 children aged 6 months to 3 years and their mothers were studied. The mothers were interviewed regarding their acceptance and perceptions about giving leafy vegetables to their young children. Their children were served a measured amount of rice and cooked leafy vegetables and mothers were asked to feed the child within about half an hour. Median intakes of leafy vegetables in children aged 6-11 months, 12-17 months and 18-35 months were 41 g, 71 g and 129 g respectively (in terms of raw green leaf). Approximately 40 g green leaf provides the recommended daily allowance for vitamin A precursors. 77% of the under-1-year-old children were breast-fed. The breast-fed children had a lower intake of vegetables than the completely weaned children. 87% of the children were found to like vegetables, 89% of the mothers liked to give vegetables to their children and 74% of the mothers answered that vegetables were good for health. Only two mothers (1.5%) refused to feed their children the leafy vegetables. The results show that leafy vegetables are acceptable to most of the mothers, and that children can eat enough leafy vegetables to meet a day's need of vitamin A precursors. Feasibility of feeding children enough green leafy vegetables at home on a regular basis needs further study.

Intake from an energy-dense porridge liquefied by amylase of germinated wheat: a controlled trial in severely malnourished children during convalescence from diarrhoea.

OBJECTIVE: To evaluate the role of an energy-dense diet liquefied with amylase-rich flour from germinated wheat (ARF) in increasing the energy intake in severely malnourished infants and young children and its acceptability to mothers. DESIGN: A randomized controlled clinical trial with two sets of controls. SETTING: Nutrition rehabilitation unit of a large diarrhoea treatment centre where mothers stay with their very severely malnourished children. SUBJECTS: 78 severely malnourished children aged 5-18 months just recovered from diarrhoea. INTERVENTION: Children were randomly assigned to receive either an energy-dense porridge made liquid by adding ARF (test diet) or an unaltered thick porridge of similar energy density (control 1 diet), or the porridge made liquid with addition of water to have the same viscosity as the test diet but of lower energy (control 2 diet), in four major meals a day for 5 days and intake was measured; breast-milk was measured by test weighing. Children also received an additional three milk-cereal meals a day. RESULTS: The mean energy intake (95% CI, P value for difference between test and control) was 385 (339-431), 289 (251-327, P < 0.005), and 255 (222-289, P < 0.001) kJ/kg.d respectively. Feeding test diet was not associated with significant adverse effects e.g. on diarrhoea, vomiting, breast-milk intake, and was well accepted by mothers. CONCLUSION: The results suggest that use of an energy-dense ARF-treated liquefied porridge increases calorie intake by very severely malnourished children during convalescence from diarrhoea, and that it does not produce any adverse effect.

A comparative pharmacokinetic study of a fixed dose combination for essential hypertensive patients: a randomized crossover study in healthy human volunteers.

BACKGROUND: This study was aimed to investigate the relative bioavailability of fixed-dose-combination (FDC) product of amlodipine, telmisartan and hydrochlorothiazide with individual marketed products in healthy male volunteers. Control of blood pressure with fixed dose combination of the above drugs acting through different mechanism have a benefit of convenient dosing in terms of compliance, lower the dose and subsequently reduce the side effects. METHODS: The authors investigated the relative bioavailability under a fasting state of the 3 drugs in a randomized, open-label, 2-treatment, 2-period, 2-sequence, crossover bioequivalence study with a washout period of 21 days. Plasma concentration of the analytes were assayed in timed samples with a simple, highly sensitive and rapid validated method using HPLC coupled to tandem mass spectrometry that had a lower limit of quantification of 1 ng/mL for all the 3 components. RESULTS: Test and reference formulations gave a mean Cmax of 5.234±0.914 ng/mL and 4.991±0.563 ng/mL, 108.839±13.601 ng/mL and 114.783±12.315 ng/mL and 97.814±10.779 ng/mL and 93.731±10.018 ng/mL for amlodipine, telmisartan and hydrochlorothiazide respectively. The AUC0-t of amlodipine, telmisartan and hydrochlorothiazide was 161.484 ng.h/mL, 1 917.644 ng.h/mL and 822.847 ng.h/mL for test formulation and 162.108 ng.h/mL, 2 014.764 ng.h/mL and 829.323 ng.h/mL for reference in the fasting state. CONCLUSION: The 90% confidence intervals for the test/reference ratio of the pharmacokinetic parameters in fasting state (mean Cmax, AUC0-t, and AUC0-∞) were within the acceptable range of 80.00-125.00. Thus, these findings clearly indicate that the FDC product is bioequivalent with the individual marketed products in terms of rate and extent of drug absorption and is well tolerated with no significant adverse reactions.

Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

Regulation of dnaB function in DNA replication in Escherichia coli by dnaC and lambda P gene products.

The dnaB protein of Escherichia coli, a multifunctional DNA-dependent ribonucleotide triphosphatase and dATPase, cross-links to ATP on ultraviolet irradiation under conditions that support rNTPase and dATPase activities of dnaB protein. The covalent cross-linking to ATP is specifically inhibited by ribonucleotides and dATP. Tryptic peptide mapping demonstrates that ATP cross-links to only the 33-kDa tryptic fragment (Fragment II) of dnaB protein. The presence of single-stranded DNA alters the covalent labeling of dnaB protein by ATP, suggesting a possible role of DNA on the mode of nucleotide binding by dnaB protein. Present studies demonstrate that the dnaC gene product binds ribonucleotides independent of dnaB protein. On dnaB-dnaC protein complex formation, covalent incorporation of ATP to dnaB protein decreases approximately 70% with a concomitant increase of ATP incorporation to dnaC protein by approximately 3-fold. The mechanism of this phenomenon has been analyzed in detail by titrating dnaB protein with increasing amounts of dnaC protein. The binding of dnaC protein to dnaB protein appears to be a noncooperative process. The lambda P protein, which interacts with dnaB protein in the bacteriophage lambda DNA replication, does not bind ATP in the presence or absence of dnaB protein. However, lambda P protein enhances the covalent incorporation of ATP to dnaB protein approximately 4-fold, suggesting a direct physical interaction between lambda P and dnaB proteins with a probable change in the modes of nucleotide binding to dnaB protein. The lambda P protein likely forms a lambda P-dnaB-ATP dead-end ternary complex. The implications of these results in the E. coli and bacteriophage lambda chromosomal DNA replication are discussed.

Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly.

The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase.

The rod outer segment ATP binding cassette (ABC) transporter protein (ABCR) plays an important role in retinal rod cells presumably transporting retinal. Genetic studies in humans have linked mutations in the ABCR gene to a number of inherited retinal diseases particularly Stargardt macular degeneration and age-related macular degeneration (ARMD). The ABCR protein is characterized by two nucleotide binding domains and two transmembrane domains, each consisting of six membrane-spanning helices. We have cloned and expressed the 376 amino acid (aa) C-terminal end of this protein (amino acid residues 1898-2273) containing the second nucleotide binding domain (NBD2) with a purification tag at its amino terminus. The expressed protein was found to be soluble and was purified using a rapid and high-yield single-step procedure. The purified protein was monomeric and migrated as a 43 kDa protein in SDS-PAGE. The purified NBD2 protein had strong ATPase activity with a K(m) of 631 microM and V(max) of 144 nmol min(-1) mg(-1). This ATPase activity on normalization was kinetically comparable to that observed for purified and reconstituted native ABCR. Nucleotide inhibition studies suggest that the binding of NBD2 is specific for ATP/dATP, and that none of the other ribonucleotides appeared to compete for binding at this site. These studies demonstrate that cloned and expressed NBD2 protein is a fully functional ATPase in the absence of the remainder of the molecule. The level of ATPase activity was comparable to that of trans-retinal-stimulated ABCR ATPase. The NBD2 expression plasmid was used to generate a Leu2027Phe mutation associated with Stargardt disease. Analysis of the ATPase activity of the mutant protein demonstrated that it had a 14-fold increase in binding affinity (K(m) = 46 microM) with a corresponding 9-fold decrease in the rate of hydrolysis (V(max) = 16.6 nmol min(-1) mg(-1)), indicating a significant alteration of the ATPase function. It also provided a molecular basis of Stargardt disease involving this mutation.

DNA helicase associated with DNA polymerase alpha: isolation by a modified immunoaffinity chromatography.

We have developed a novel immunoaffinity method for isolating a DNA polymerase alpha-associated DNA helicase from the yeast Saccharomyces cerevisiae. Earlier we have reported the characterization of a DNA helicase activity associated with the multiprotein DNA polymerase alpha complex from yeast [Biswas, E. E., Ewing, C. M., & Biswas, S. B. (1993) Biochemistry 32, 3030-3027]. We report here the isolation of the DNA helicase from the DNA polymerase alpha (pol alpha) complex bound to an anti-pol alpha immunoaffinity matrix. The DNA helicase activity eluted at approximately 0.35 M NaCl concentration. The eluted ATPase/helicase peak was further purified by size-exclusion high-performance liquid chromatography (HPLC). At low ionic strength (50 mM NaCl), it remained associated with other proteins and eluted as a large polypeptide complex. At high ionic strength (500 mM NaCl), the helicase dissociated, and the eluted ATPase/helicase fraction contained 90-, 60-, and 50-kDa polypeptides. Photoaffinity cross-linking of helicase with ATP during the isolation process demonstrated a 90-kDa polypeptide to be the likely ATP binding component of the helicase protein. The DNA helicase has single-stranded DNA (ssDNA)-stimulated ATPase and dATPase activities. The ATPase activity was stimulated by yeast replication protein A (RPA). The DNA helicase activity was stimulated by Escherichia coli ssDNA binding protein and RPA. The DNA helicase migrated on a DNA template in the 5'-->3' direction which is also the overall direction of migration of pol alpha on the lagging strand of the replication fork.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the DNA-dependent ATPase and a DNA unwinding activity associated with the yeast DNA polymerase alpha complex.

We have analyzed the ATPase and dATPase activities associated with the yeast DNA polymerase alpha complex. The ATPase/dATPase was primarily a single-stranded DNA-dependent ATPase. Analysis of the stimulatory effect of a large number of DNA substrates demonstrated that polynucleotides longer than 60 nucleotides (nts) had the maximal effect. The stimulation by oligonucleotides smaller than 60 nts, in general, decreased proportionally with decreased length of the oligomer. Poly- or oligopyrimidines were twice as stimulatory as the poly- or oligopurines of the same length. In addition to DNA, replication protein A (RP-A), a single-stranded DNA (ssDNA) binding protein, also stimulated the ATPase activity. Photo-cross-linking of the ATP binding component of the pol alpha complex to [alpha-32P]ATP at 0 degree C resulted in the exclusive labeling of a 90-kDa polypeptide. The labeling was inhibited by ATP and dATP but not by any other ribo- or deoxynucleotides, which suggest that the 90-kDa polypeptide is specific for ATP/dATP binding and possibly the active site for the ATPase/dATPase. We have also reported here a novel DNA unwinding activity associated with the multiprotein complex of DNA polymerase alpha. The complex was able to unwind M13mp19 ssDNA hybridized to an oligonucleotide (17-60 nucleotides long) with a protruding 3'-terminus. Regardless of the size of the duplex, the DNA unwinding was significantly stimulated by RP-A, while RP-A itself did not have any DNA unwinding activity. Consequently, it appeared that the DNA polymerase alpha complex possessed a putative RP-A-dependent helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity.

We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities. Highly purified tryptic fragments containing various domains of DnaB helicase were prepared. Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase. Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I. The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail. The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished. Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III. Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities. Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies. SE-HPLC analysis indicated that fragment II had an apparent hexameric form. However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired. Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A.

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product.

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein.

The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB X dnaC protein complex has been studied by using fluorescent nucleotide analogues. Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues. Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 microM) and a 8.5-fold enhancement of fluorescence emission of the nucleotide. Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer. TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein. TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity. Formation of dnaB X dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity. Kinetic analysis of the ATPase activity of dnaB X dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax). The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA. The 2'-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.