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At least 649 records · Page 36Linked to original sources
PubMed2005

Large scale molecular dynamics simulation of native and mutant dihydropteroate synthase-sulphanilamide complexes suggests the molecular basis for dihydropteroate synthase drug resistance.

Antibiotic resistance is hampering the efficacy of drugs in the treatment of several pathological infections. Dihydropteroate synthase (DHPS) has been targeted by sulphonamide inhibitors for the past 60 years and has developed different amino acid mutations to survive sulpha drug action. We couple homology modelling techniques and massively parallel molecular dynamics simulations to study both the drug-bound and apo forms of native and mutant DHPS. Simulations of the complex between sulphanilamide and Streptomyces pneumoniae, DHPS shows how sulphanilamide is able to position itself close to 6-hydroxymethyl-7, 8-dihydropteridine-phosphate in a suitable position for the enzymatic transformation whereas in the mutant complex the sulpha drug is expelled from the catalytic site. Our simulations, therefore, provide insight into the molecular basis for drug resistance with S. pneumoniae DHPS.

Anti-Bacterial Agents↗
PubMed2006

The hypolipidemic agent guggulsterone regulates the expression of human bile salt export pump: dominance of transactivation over farsenoid X receptor-mediated antagonism.

Conversion of cholesterol to bile acids in the liver is initiated by the rate-limiting enzyme cholesterol 7alpha-hydroxylase (CYP7A1) and excretion of bile acids from the liver is mediated by the bile salt export pump (BSEP). The expression of CYP7A1 and BSEP is coordinately regulated by a negative feedback and positive feed-forward mechanism, respectively, through bile acid-mediated activation of farsenoid X receptor (FXR). It is well established that hypolipidemic agent guggulsterone is an FXR antagonist and down-regulates FXR target genes. In this study, however, we have demonstrated that guggulsterone synergistically induced the expression of BSEP in cells treated with FXR agonist bile acids. A dissection study located in the BSEP promoter an activating protein (AP)-1 site supporting the action of guggulsterone. Deletion or mutation of the AP-1 element was diminished, whereas insertion of the AP-1 element into a heterologous promoter enhanced activation of the promoter by guggulsterone. Selective c-Jun N-terminal kinase and extracellular signal-regulated kinase inhibitors markedly decreased the transactivation, suggesting an involvement of AP-1 activation pathway in the up-regulation of BSEP by guggulsterone. Consistent with its FXR antagonism, guggulsterone antagonized bile acid-mediated transactivation of BSEP promoter when the AP-1 element was disrupted. In conclusion, guggulsterone regulates BSEP expression through composite mechanisms, and the transactivation through the AP-1 element is dominant over the FXR-mediated antagonism. The up-regulation of BSEP expression by guggulsterone without activating FXR pathway as an FXR agonist to suppress CYP7A1 expression represents a possible mechanism for guggulsterone-mediated hypolipidemic effect.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Effects of kinesin mutations on neuronal functions.

Kinesin is believed to generate force for the movement of organelles in anterograde axonal transport. The identification of genes that encode kinesin-like proteins suggests that other motors may provide anterograde force instead of or in addition to kinesin. To gain insight into the specific functions of kinesin, the effects of mutations in the kinesin heavy chain gene (khc) on the physiology and ultrastructure of Drosophila larval neurons were studied. Mutations in khc impair both action potential propagation in axons and neurotransmitter release at nerve terminals but have no apparent effect on the concentration of synaptic vesicles in nerve terminal cytoplasm. Thus kinesin is required in vivo for normal neuronal function and may be active in the transport of ion channels and components of the synaptic release machinery to their appropriate cellular locations. Kinesin appears not to be required for the anterograde transport of synaptic vesicles or their components.

Action Potentials↗
PubMed1991

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.

Animals↗
PubMed2002

Exendin-4 as a stimulator of rat insulin I gene promoter activity via bZIP/CRE interactions sensitive to serine/threonine protein kinase inhibitor Ro 31-8220.

Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic beta-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a -410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative G alpha(s), was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 cAMP-regulated guanine nucleotide exchange factor (Epac2) failed to alter the response to Ex-4. Ro 31-8220, a serine/threonine protein kinase inhibitor that targets PKC as as well as the 90-kDa ribosomal S6 kinase (RSK) and mitogen- and stress-activated protein kinase (MSK) family of cAMP response element-binding protein (CREB) kinases, blocked the stimulatory action of Ex-4 at RIP1-Luc. However, selective inhibition of PKC using K-252c, prolonged exposure to phorbol 1,2-myristate-13-acetate, or cotransfection with dominant-negative atypical PKC-zeta, was without effect. A-CREB, a dominant-negative inhibitor of basic region-leucine zipper transcription factors (bZIPs) related in structure to CREB, inhibited the action of Ex-4 at RIP1-Luc, whereas A-ATF-2 was ineffective. Similarly, introduction of deletions at the RIP1 cAMP response element (CRE), or truncation of RIP1 to remove the CRE, nearly abolished the action of Ex-4. Inactivating mutations introduced at the A4/A3 elements, binding sites for the glucose-regulated homeodomain transcription factor PDX-1, did not diminish the response to Ex-4, although a marked reduction of basal promoter activity was observed. The glucose-dependent stimulation of RIP1-Luc by Ex-4 was reproduced using a synthetic reporter (RIP1-CRE-Luc) incorporating multimerized CREs of the RIP1 nonpalindromic sequence 5'-TGACGTCC-3'. It is concluded that the bZIP and CRE-mediated stimulation of RIP1 by Ex-4 explains, at least in part, how this insulinotropic hormone facilitates transcriptional activity of the rat insulin I gene.

Animals↗
PubMed2005

Molecularly-targeted therapies for non-small cell lung cancer.

Targeting cell-signalling pathways that confer survival advantage to cancer cells has become a major focus of investigation for the treatment of various malignancies. Non-small cell lung cancer (NSCLC), a disease with wide molecular heterogeneity, has become a main testing ground for the evaluation of various targeted agents. Inhibition of the epidermal growth factor pathway with erlotinib results in improved survival and symptom control for patients with advanced NSCLC who progressed following one or two prior chemotherapy regimens. Gefitinib, the first epidermal growth factor receptor (EGFR) inhibitor to be approved by the FDA, failed to demonstrate survival advantage over placebo in a large Phase III trial for patients with advanced NSCLC. The results of this study have raised several important clinical and biological issues that may be relevant for the development of other targeted agents. Recent identification of mutations in the ATP-binding pocket of the EGFR is the first step towards proper patient selection for therapy with an EGFR tyrosine kinase inhibitor. In addition, predictive potential has also been seen with EGFR gene amplification. It is unclear whether monoclonal antibodies against the EGFR may be active independent of the EGFR mutation, as the site of action is different from tyrosine kinase inhibitors. A recent randomised clinical trial that combined the antiangiogenic agent bevacizumab with chemotherapy has demonstrated survival advantage over chemotherapy alone for certain subsets of patients with advanced NSCLC. The exciting results of this study represent an important advance in the treatment of patients with advanced NSCLC.

Animals↗
PubMed2005

Possible relationship between elevated plasma ACTH and tall stature in familial glucocorticoid deficiency.

Familial glucocorticoid deficiency (FGD) is characterized clinically by severe glucocorticoid deficiency associated with failure of adrenal responsiveness to ACTH but not with mineralcorticoid deficiency. Excessive growth was described previously in some patients with FGD, many of whom were shown to have mutations in the ACTH receptor gene. The mechanisms responsible for their excessive growth are unknown. We analyzed the ACTH receptor gene in three patients with FGD and discussed the causes of excessive growth in FGD. No mutations were detected in the coding and promoter regions of the ACTH receptor gene of one female patient who had tall stature (+ 2.41S.D.) and advanced bone age (10 years 9 months) when she was 4 years 9 months old. Her plasma ACTH level had been elevated until then (124-2,684 pg/ml). Moreover, plasma estradiol was elevated for her age (21.3 pg/ml), and it decreased in response to the dexamethasone suppression test (from 25.4 to 6.9 pg/ml). Elevated plasma estradiol was apparently related to the increase in plasma ACTH and played a major role in excessive growth in this patient. On the other hand, the genetic analysis showed that the other two patients who were siblings were homozygous for the R137W mutation. Clinically, they responded well to hydrocortisone replacement therapy with almost normal plasma ACTH levels. Although all patients with the R137W mutation reported previously were tall, our patients were of normal height. We speculate that the major causes of excessive growth in FGD are not only from ACTH receptor mutation, but also from the action of elevated plasma ACTH.

Adolescent↗
PubMed2004

The Vif protein of human immunodeficiency virus type 1 (HIV-1): enigmas and solutions.

HIV-1 and other complex retroviruses express six auxiliary genes in addition to the canonical retroviral genes, gag, pol and env. Vif (virion infectivity factor) protein is absolutely essential for productive HIV-1 infection of peripheral blood lymphocytes and macrophages, the two major HIV-1 target cells in vivo. However, Vif is not required for production of infectious particles in several human cell lines. In spite of the prominent phenotype of Vif mutations, the mechanism of its action remains unknown. During the last decade several models were suggested to explain the mechanism of Vif activity. One view holds that Vif is active in virions after budding or after entry into target cells during the early stages of HIV-1 replications. The second view places the action of Vif at the late stage of HIV-1 replication in virus producing cells, which affects the production of infectious virus. According to this view, Vif either compensates the cell factor required for production of infectious virus, or alternatively, it neutralizes a cell factor, which prevents the production of infectious particles in these cells. This review is addressed to summarize the models envisioned to explain Vif activities. The findings described here, that Vif interacts with viral and cellular components, elaborates the importance of Vif as a novel target for developing anti HIV-1 drugs.

Gene Products, vif↗
PubMed1993

The P1 plasmid partition complex at parS. II. Analysis of ParB protein binding activity and specificity.

The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF). ParB binds to regions of parS that flank the IHF binding site. In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns. Methylation protection and interference experiments were performed on supercoiled plasmids. Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS. These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS. ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity. The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone. The stimulation by IHF is strongly influenced by helical phasing. These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.

Bacterial Proteins↗
PubMed2003

The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subunits.

BACKGROUND: Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the gamma-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the alpha1, alpha 2, or beta 2 subunits. METHODS: GABAA receptor alpha 1 or alpha 2, beta 2 or beta 3, and gamma 2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type alpha 1 beta 2 gamma 2s and alpha 2 beta 3 gamma 2s receptors, and in receptors harboring mutations in TM2, such as alpha1(S270W)beta 2 gamma 2s, alpha 1 beta 2(N265W)gamma 2s, and alpha2(S270I)beta 3 gamma 2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl- currents were compared in the wild-type and mutant GABAA receptors. RESULTS: Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA alpha 1 beta 2 gamma 2s receptor and alpha 2 beta 3 gamma 2s receptor. Substitution of Ser270 in TM2 of the alpha subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in alpha1(S270W)beta 2 gamma 2s and alpha 2(S270I)beta 3 gamma 2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the beta subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. CONCLUSIONS: Positions Ser270 of the GABAA alpha1 and alpha2 subunits, but not Asn265 in the TM2 of the beta2 subunit, are critical for regulation of the GABAA receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the alpha subunit of the GABAA receptor.

Amino Acid Sequence↗
PubMed2000

High salivary acetaldehyde after a moderate dose of alcohol in ALDH2-deficient subjects: strong evidence for the local carcinogenic action of acetaldehyde.

BACKGROUND: Due to a point mutation, aldehyde dehydrogenase-2 (ALDH2) isoenzyme is deficient in 30% to 50% of Asians. Among Asian ALDH2-deficient heavy drinkers, the risk for digestive tract cancers is markedly increased (odds ratio 3.4-54.2). The reason for this is unknown but could be due to the local carcinogenic action of acetaldehyde. METHODS: Salivary and blood acetaldehyde levels were determined in 20 healthy Asians after a moderate dose of alcohol (0.5 g/kg of body weight). Salivary acetaldehyde production capacity from ethanol in vitro was measured also. ALDH2 genotype of the Asians was determined from isolated leukocyte-deoxyribonucleic acid by polymerase chain reaction/restriction fragment length polymorphism method. Acetaldehyde content of parotid gland saliva was measured in three ALDH2-deficient Asians and three White subjects with normal ALDH2 after the same dose of ethanol. RESULTS: Seven of the Asians were heterozygous for the mutant ALDH2*2 allele (flushers). They had two to three times higher salivary acetaldehyde levels than the Asians (n = 13) with normal ALDH2 throughout the follow-up period of 240 min (p < 0.001). Only in the flushers did the parotid gland contribute to salivary acetaldehyde production. The in vitro capacity of saliva to produce acetaldehyde from ethanol was equal in both groups. The flushers' blood acetaldehyde levels were only one ninth of the levels in saliva. CONCLUSIONS: By using this human "knockout model" for deficient acetaldehyde removal, we found that in addition to oral microflora, acetaldehyde in saliva may also originate from the oxidation of ethanol in the parotid gland. When combined with earlier epidemiological data, these results offer a strong evidence for the local carcinogenic action of acetaldehyde in humans.

Acetaldehyde↗
PubMed2004

MutS inhibits RecA-mediated strand exchange with platinated DNA substrates.

Human cell lines and Escherichia coli dam mutants are sensitive to the cytotoxic action of the anticancer agent, cisplatin. Introduction of mutations disabling DNA mismatch repair into these cell lines renders them resistant to the action of this drug. We used RecA-mediated strand exchange between homologous phiX174 molecules, one that was platinated and the other that was unmodified, to show that strand transfer is decreased in a dose-dependent manner. Transfer was severely decreased at 10 adducts per molecule (5,386 bp) and abolished with 24 adducts. At low levels of adduction, addition of MutS to the reaction further decreases the rate and yield in a dose-dependent manner. MutL addition was without effect even in the presence of MutS. The results suggest that although mismatch repair is beneficial for mutation avoidance, its antirecombination activity on inappropriate substrates can be lethal to the cell.

Adenosine Triphosphatases↗
PubMed2003

Mutations in cardiac sodium channels: clinical implications.

Voltage-gated sodium channels (VGSCs) are critical transmembrane proteins responsible for the rapid action potential upstroke in most excitable cells. Recently discovered mutations in VGSCs, which underlie idiopathic clinical disease, have emphasized the importance of these channels in tissues such as skeletal muscle, nervous system, and myocardium. Mutations in the gene encoding the cardiac sodium channel isoform (SCN5A) have been linked to at least three abnormal phenotypes: variant 3 of the Long QT syndrome (LQT-3); Brugada's syndrome (BrS); and isolated cardiac conduction disease (ICCD). Mutations in SCN5A manifest as one or more of these clinical phenotypes - the precise distinction between these diseases is increasingly subtle. Clinical management of LQT-3 and diagnosis of BrS with the local anesthetic flecainide has proven promising. Channels associated with LQT-3 (D1790G) and BrS (Y1795H) both show more sensitivity to flecainide than wild-type (WT) channels, while lidocaine sensitivity is unchanged. One plausible explanation for differential drug sensitivity is that mutant channels may allow more access to a receptor site compared with WT through altered protein allosteric changes during an action potential. The high affinity binding site for local anesthetic block has been identified in the pore region of the channel. This region is not water accessible during the closed state, thus requiring channel opening for charged drug (flecainide and mexiletine) access and block. Channel mutations which disrupt inactivation biophysics lead to increased drug binding by altering the time the binding site is accessible during an action potential. Neutral drugs (lidocaine) which are not dependent on channel opening for binding site access will not be sensitive to mutations that alter channel inactivation properties. Interestingly another LQT-3 mutant (Y1795C) shows no change in flecainide sensitivity, suggesting that although drug effects of SCN5A mutations cross disease boundaries, clinical management with flecainide will be beneficial to patients in a mutation-specific manner.

Animals↗
PubMed1987

A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin.

Primer-directed mutagenesis was employed to introduce an A2058----G transition in plasmid-encoded Escherichia coli 23S RNA at a site that has been implicated, indirectly, in erythromycin binding. The mutation raises the growth tolerance of cells from 30 to 300 micrograms/ml of erythromycin, and cells grown in the presence of erythromycin contain ribosomes with high levels of mutated 23S RNA. In these cells, wild type 50S subunits 'fall off' the message and are selectively degraded, possibly as a result of an erythromycin-induced conformational change. A fast in vitro poly(U) assay revealed minimal effects of erythromycin on elongation beyond tetrapeptides. We correlated these results with the literature data and concluded that erythromycin acts immediately post-initiation and directly, or indirectly, destabilizes mRNA-bound 70S ribosomes, and prevents their recycling by causing 50S subunit degradation.

Autoradiography↗
PubMed1996

Cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation.

The transposase protein encoded by the insertion element IS903 belongs to an unusual class of DNA-binding proteins, termed cis-acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine-to-proline substitution at amino acid 25 (T25P) reduced cis preference about 60-fold. The half-life of this mutant transposase was significantly longer than that of the wild-type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.

DNA Nucleotidyltransferases↗
PubMed1999

The Ras mutant D119N is both dominant negative and activated.

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

3T3 Cells↗
PubMed1978

A site of action for tRNA mediated regulation of the ilvOEDA operon of Escherichia coli K12.

Transfer RNA (tRNA), rho factor threonine deaminase and the ilvO locus are molecular participants in the regulation of isoleucine-valine (ilv) biosynthesis. Isogenic strains have been constructed with the hisT76 mutation in pairwise combination with ilvO mutations, the rho221 mutation and the ilvDAC115 deletion mutation. The role of the altered tRNA of the hisT76 mutation was found to be independent of the sites of action of the ilvO- mutation, rho factor, and threonine deaminase. The expression of the ilvOEDA operon is stimulated 2-fold when the hisT76 mutation is present in strains containing either ilvO- or rho221 mutations. The expression of the ilvOEDA operon remains nonrepressed in a hisT76 strain deleted for threonine deaminase. These results indicate that the hisT76 undermodified tRNAs are influencing the initiation of transcription of the ilvOEDA operon.

Escherichia coli↗
PubMed1994

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.

Adenosine Triphosphatases↗