DNA damage recognition and nucleotide excision repair in mammalian cells.
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Animals↗
Biomedical subjects
E Evans
Publications and source records attributed to E Evans.
Roles for mismatch repair factors in regulating genetic recombination.
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Animals↗
Effect of maternal nutrition on brown adipose tissue and its prolactin receptor status in the fetal lamb.
We investigated the influence of maternal nutritional enhancement during the second half of gestation on prolactin receptor (PRLR) abundance in fetal brown adipose tissue (BAT) and liver close to term (i.e. 141-144 d gestation). Ewes were provided with 100% (i.e. control; n = 8) or 150% (i.e. well-fed; n = 7) of their metabolic requirements from 80 to 144 d gestation. Crude plasma membranes were prepared from fetal BAT and hepatic tissue, and individual molecular weight isoforms for the long and short forms of the PRLR were detected by immunoblotting. Mitochondrial preparations were prepared from BAT to measure the amount of the BAT-specific mitochondrial uncoupling protein-1 and its thermogenic activity (i.e. guanosine 5'-diphosphate binding). Fetuses sampled from well-fed ewes were heavier (controls, 3927 +/- 196 g; well-fed, 4783 +/- 219 g; p = 0.01) but possessed less BAT per kilogram body weight (controls, 5.92 +/- 0.43 g/kg; well-fed, 3.85 +/- 0.19 g/kg; p = 0.001), which had a greater uncoupling protein-1 abundance (controls, 56 +/- 5% of reference; well-fed, 78 +/- 9% of reference; p < 0.01) and higher thermogenic activity (controls, 157 +/- 41 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; well-fed, 352 +/- 36 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; p < 0.01) than controls. Multiple isoforms of the long and short forms of the P1LR were detected in all tissues. BAT from well-fed fetuses had a higher abundance of the 15-kD isoform of the long form of the PRLR (controls, 1.6 +/- 0.4 densitometric units; well-fed, 16.3 +/- 2.0 densitometric units; p < 0.001). This isoform was not detected in hepatic tissue. Maternal nutrient intake had no effect on any other isoforms of the PRLR in BAT or liver. In conclusion, increasing the quantity of feed provided in late gestation acts to promote fetal weight and BAT maturation, the combination of which will enhance neonatal viability.
Adipose Tissue, Brown↗
Looking inside molecular bonds at biological interfaces with dynamic force spectroscopy.
Weak non-covalent interactions between large molecules govern interfacial structure and adhesion in biology. Because of thermal activation, these bonds have modest lifetimes and bond lifetimes are progressively shortened under application of external force. Theory predicts that bond survival time depends on how fast the force is applied and the expected survival time specifies the most likely breakage force (strength) at a given loading rate (force/time). Plotted as a function of log(e) (loading rate), the dynamic spectrum of bond strength provides an image of the prominent barriers traversed in the energy landscape along the unbinding pathway, which establishes a direct link between measurements of bond force and molecular-scale chemistry. Experimentally, the challenge is to measure bond strength over several orders of magnitude in loading rate. With a recently designed probe technique, we have measured strengths of single receptor-ligand bonds and receptor-membrane anchoring over an enormous range of loading rates from 10(-1) pN/s to 10(5) pN/s, which reveals an inner view of the complexity of these interactions.
Biotin↗
Conserved residues of human XPG protein important for nuclease activity and function in nucleotide excision repair.
The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.
Amino Acid Sequence↗
Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy.
Atomic force microscopy (AFM) has been used to measure the strength of bonds between biological receptor molecules and their ligands. But for weak noncovalent bonds, a dynamic spectrum of bond strengths is predicted as the loading rate is altered, with the measured strength being governed by the prominent barriers traversed in the energy landscape along the force-driven bond-dissociation pathway. In other words, the pioneering early AFM measurements represent only a single point in a continuous spectrum of bond strengths, because theory predicts that these will depend on the rate at which the load is applied. Here we report the strength spectra for the bonds between streptavidin (or avidin) and biotins-the prototype of receptor-ligand interactions used in earlier AFM studies, and which have been modelled by molecular dynamics. We have probed bond formation over six orders of magnitude in loading rate, and find that the bond survival time diminished from about 1 min to 0.001 s with increasing loading rate over this range. The bond strength, meanwhile, increased from about 5 pN to 170 pN. Thus, although they are among the strongest noncovalent linkages in biology (affinity of 10(13) to 10(15) M(-1)), these bonds in fact appear strong or weak depending on how fast they are loaded. We are also able to relate the activation barriers derived from our strength spectra to the shape of the energy landscape derived from simulations of the biotin-avidin complex.
Biotin↗
Strength of a weak bond connecting flexible polymer chains.
Bond dissociation under steadily rising force occurs most frequently at a time governed by the rate of loading (Evans and Ritchie, 1997 Biophys. J. 72:1541-1555). Multiplied by the loading rate, the breakage time specifies the force for most frequent failure (called bond strength) that obeys the same dependence on loading rate. The spectrum of bond strength versus log(loading rate) provides an image of the energy landscape traversed in the course of unbonding. However, when a weak bond is connected to very compliant elements like long polymers, the load applied to the bond does not rise steadily under constant pulling speed. Because of nonsteady loading, the most frequent breakage force can differ significantly from that of a bond loaded at constant rate through stiff linkages. Using generic models for wormlike and freely jointed chains, we have analyzed the kinetic process of failure for a bond loaded by pulling the polymer linkages at constant speed. We find that when linked by either type of polymer chain, a bond is likely to fail at lower force under steady separation than through stiff linkages. Quite unexpectedly, a discontinuous jump can occur in bond strength at slow separation speed in the case of long polymer linkages. We demonstrate that the predictions of strength versus log(loading rate) can rationalize conflicting results obtained recently for unfolding Ig domains along muscle titin with different force techniques.
Animals↗
Why should obesity be managed? The obese individual's perspective.
Weight loss can be achieved using a variety of different methods, alone or in combination, including energy restricted diets, drug treatment and surgical intervention. The difficulty is maintaining weight loss over a prolonged period. Physicians, dietitians and nurses are often pessimistic about their ability to manage obesity. Such negative attitudes, combined with the erroneous belief that obesity is not a serious medical condition, have adversely affected the level of care received by obese patients. Despite this, the family doctor is often approached for help with weight control. Obese patients are generally well informed about diet and weight issues, and are in a good position to critically assess the weight loss advice given by their doctor. The perception of such advice formed part of a questionnaire completed by obese people (346 female/24 male) who successfully reduced their weight while attending a weight loss group. Eighty per cent had previously been advised by their doctor to lose weight, but guidance on how to do this was generally judged to be poor. Only 22% of subjects received positive advice, although 23% of subjects reported that their doctor's advice was indirectly responsible for their weight loss. Patients derived evident health benefits from their weight loss and were generally given a positive response on returning to their doctor. To help patients lose weight, doctors must realise that obesity is a serious chronic medical condition. Ongoing help and support from doctors and other healthcare professionals is a key element in successful long-term weight management.
Attitude of Health Personnel↗
Spinal clonidine prolongs labor analgesia from spinal sufentanil and bupivacaine.
UNLABELLED: We sought to determine whether spinal clonidine 50 microg prolongs the analgesia from the spinal administration of sufentanil 7.5 microg and bupivacaine 2.5 mg early in the first stage of labor. Thirty patients were randomized to receive a 2-mL spinal injection of sufentanil 7.5 microg + bupivacaine 2.5 mg with or without clonidine 50 microg using a combined spinal-epidural (CSE) technique. Pain, nausea, pruritus, sedation, motor block, blood pressure, and heart rate were assessed until the patient requested additional analgesia. Analgesia was significantly prolonged in patients who received spinal sufentanil + bupivacaine + clonidine (197 +/- 70 vs 132 +/- 39 min; P = 0.004). Pain scores and side effects, including motor block, sedation, and hypotension, were similar between groups. Spinal clonidine significantly prolongs labor analgesia from spinal sufentanil and bupivacaine without producing serious adverse side effects. IMPLICATIONS: We studied the effects of spinal clonidine administered with spinal sufentanil and bupivacaine on labor analgesia using a combined spinal-epidural technique and conclude that spinal clonidine significantly prolongs labor analgesia from spinal sufentanil and bupivacaine without producing serious adverse effects.
Adult↗
Day case surgery has benefited patients with cataract in North Gwent.
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Ambulatory Surgical Procedures↗
Energy landscapes of biomolecular adhesion and receptor anchoring at interfaces explored with dynamic force spectroscopy.
Beyond covalent connections within protein and lipid molecules, weak noncovalent interactions between large molecules govern properties of cellular structure and interfacial adhesion in biology. These bonds and structures have limited lifetimes and so will fail under any level of force if pulled on for the right length of time. As such, the strength of interaction is the level of force most likely to disrupt a bond on a particular time scale. For instance, strength is zero on time scales longer than the natural lifetime for spontaneous dissociation. On the other hand, if driven to unbind or change structure on time scales shorter than needed for diffusive relaxation, strength will reach an adiabatic limit set by the maximum gradient in a potential of mean force. Over the enormous span of time scales between spontaneous dissociation and adiabatic detachment, theory predicts that bond breakage under steadily rising force occurs most frequently at a force determined by the rate of loading. Moreover, the continuous plot (spectrum) of strength expressed on a scale of loge(loading rate) provides a map of the prominent barriers traversed in the energy landscape along the force-driven pathway and reveals the differences in energy between barriers. Illustrated with results from recent laboratory measurements, dynamic strength spectra provide a new view into the inner complexity of receptor-ligand interactions and receptor lipid anchoring.
Animals↗
Surface-catalysed disinfection of thick Pseudomonas aeruginosa biofilms.
Transition metal catalysts were incorporated into polymers which formed the surface for bacterial attachment and biofilm formation in a constant depth film fermenter (100 microns thickness), flow chamber (about 30 microns thickness) and in batch culture (< 30 microns thickness). The catalysts drive the breakdown of persulphates to reactive oxygen species. When Pseudomonas aeruginosa biofilms were exposed to dilute solutions of potassium monopersulphate (20 micrograms ml-1-1 mg ml-1), significant enhancement of killing was notable for catalyst-containing surfaces over that of controls. The degree of enhancement was greatest for thin films, but was nevertheless significant for the 100 microns thick biofilms. Fluorescence probes and viability staining, in conjunction with laser confocal microscopy, showed that reactive species were generated at the biofilm-substratum interface and killed the biofilm from the inside. Reaction-diffusion limitation now concentrates the active species within the biofilm rather than protecting it, and a diffusion bump is established whereby further treatment agent is drawn to the substratum enabling relatively thick biofilms to be disinfected.
Biofilms↗
Newly arising fibroadenomas in women aged 35 and over.
BACKGROUND: Fifty-one cases of de novo fibroadenoma in women aged 35 years and older were found during an analysis of 117,729 visits to the Wesley Breast Clinic from 1990 to 1996. METHODS: The clinical, mammographic and ultrasound diagnosis of fibroadenoma was confirmed by either fine needle aspiration cytology or histology of an open biopsy specimen. In all cases there was a well-documented previous visit available for review, at which there was no clinical or radiological evidence of the fibroadenoma. RESULTS: Thirty-seven of the de novo fibroadenomas were palpable, the remainder satisfying strict mammographic and/or ultrasound criteria. Four of the new fibroadenomas were in women aged 50-52. CONCLUSIONS: This study provides information about the natural history of fibroadenomas, confirming that they can appear for the first time in middle-aged women. This has important clinical implications, since new lesions appearing in women over 35 have tended to be automatically categorized as suspicious of carcinoma. However, a multidisciplinary approach involving clinical examination, mammography, ultrasound, and fine needle aspiration cytology or core biopsy can result in a confident diagnosis of fibroadenoma. This will allow some women with new lesions to be managed conservatively rather than by open biopsy.
Adult↗
Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.
During nucleotide excision repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core nucleotide excision repair factors. Opening is followed by dual incision, with the 3' cleavage made first.
Adenosine Triphosphate↗
Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.
Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.
Adenosine Triphosphate↗
Dynamic strength of molecular adhesion bonds.
In biology, molecular linkages at, within, and beneath cell interfaces arise mainly from weak noncovalent interactions. These bonds will fail under any level of pulling force if held for sufficient time. Thus, when tested with ultrasensitive force probes, we expect cohesive material strength and strength of adhesion at interfaces to be time- and loading rate-dependent properties. To examine what can be learned from measurements of bond strength, we have extended Kramers' theory for reaction kinetics in liquids to bond dissociation under force and tested the predictions by smart Monte Carlo (Brownian dynamics) simulations of bond rupture. By definition, bond strength is the force that produces the most frequent failure in repeated tests of breakage, i.e., the peak in the distribution of rupture forces. As verified by the simulations, theory shows that bond strength progresses through three dynamic regimes of loading rate. First, bond strength emerges at a critical rate of loading (> or = 0) at which spontaneous dissociation is just frequent enough to keep the distribution peak at zero force. In the slow-loading regime immediately above the critical rate, strength grows as a weak power of loading rate and reflects initial coupling of force to the bonding potential. At higher rates, there is crossover to a fast regime in which strength continues to increase as the logarithm of the loading rate over many decades independent of the type of attraction. Finally, at ultrafast loading rates approaching the domain of molecular dynamics simulations, the bonding potential is quickly overwhelmed by the rapidly increasing force, so that only naked frictional drag on the structure remains to retard separation. Hence, to expose the energy landscape that governs bond strength, molecular adhesion forces must be examined over an enormous span of time scales. However, a significant gap exists between the time domain of force measurements in the laboratory and the extremely fast scale of molecular motions. Using results from a simulation of biotin-avidin bonds (Izrailev, S., S. Stepaniants, M. Balsera, Y. Oono, and K. Schulten. 1997. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys. J., this issue), we describe how Brownian dynamics can help bridge the gap between molecular dynamics and probe tests.
Avidin↗
The vaccinia virus I1 protein is essential for the assembly of mature virions.
The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.
Amino Acid Sequence↗
Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.
Nucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causative mutations and strongly reduced levels of encoded protein were identified in XP-F patients. The XPF protein was purified from mammalian cells in a tight complex with ERCC1. This complex is a structure-specific endonuclease responsible for the 5' incision during repair. These results demonstrate that the XPF, ERCC4, and ERCC11 genes are equivalent, complete the isolation of the XP genes that form the core nucleotide excision repair system, and solve the catalytic function of the XPF-containing complex.
Animals↗