The analysis of oral air using selected ion flow tube mass spectrometry in persons with and without a history of oral malodour.

OBJECTIVES: Oral malodour is a common disorder predominantly caused by bacterial metabolism of food stuffs in the mouth. It is routinely diagnosed and monitored by either the subjective rating or the measurement of oral volatile sulphur compound (VSC) levels. Non-sulphur compounds are also believed to contribute significantly to the condition although there is currently no direct means to assess their levels. In this study, we utilized selective flow tube mass spectrometry (SIFT-MS) to measure, in real time, a range of sulphur and non-sulphur containing compounds in oral air to determine whether the technique can be used to objectively monitor oral malodour. METHODS: Oral malodour was assessed using organoleptic scores in subjects with and without a history of oral malodour (n = 18) by a trained rater, while the chemical composition of oral air was analysed by both VSC sensor and SIFT-MS. RESULTS: Total VSC levels were significantly correlated with levels of hydrogen sulphide and methylmercaptan measured by SIFT-MS, but not with organoleptic scores. In subjects with elevated organoleptic score, only levels of methylmercaptan were significantly elevated. In three subjects with elevated tongue organoleptic scores but normal total VSC levels, SIFT-MS suggested that one subject possessed high levels of oral acetone while another had high oral levels of acetic acid. CONCLUSIONS: Our data suggest that SIFT-MS can be used to assess a wide range of compounds in oral air in addition to VSC to provide a clearer picture of the chemical nature of malodour. This may assist in the diagnosis and monitoring of the condition.

Mutations in the glucocerebrosidase gene are associated with early-onset Parkinson disease.

OBJECTIVE: To evaluate the frequency of glucocerebrosidase (GBA) mutations in cases and controls enrolled in the Genetic Epidemiology of Parkinson's Disease (GEPD) study. METHODS: We sequenced all exons of the GBA gene in 278 Parkinson disease (PD) cases and 179 controls enrolled in GEPD, with a wide range of age at onset (AAO), and that included a subset of 178 Jewish cases and 85 Jewish controls. Cases and controls were recruited without knowledge of family history of PD, and cases were oversampled in the AAO < 50 years category. RESULTS: 13.7% of PD cases (38/278) carried GBA mutations, compared with 4.5% of controls (8/179) (odds ratio [OR] 3.4, 95% CI 1.5 to 7.4). The frequency of GBA mutations was 22.2% in 90 cases with AAO < or = 50 years, compared with 9.7% in 185 cases with AAO > 50 years (OR 2.7, 95% CI 1.3 to 5.3). Adjusting for age at the time of evaluation, sex, family history of PD, and Jewish ancestry, GBA carriers had a 1.7-year-earlier AAO of PD (95% CI 0.5 to 3.3, p < 0.04) than noncarriers. The average AAO of PD was 2.5 years earlier in carriers with an AAO < or = 50 years compared with noncarriers (95% CI 0.6 to 4.5, p < 0.01) and this was not seen in the AAO > 50 years group. The frequency of GBA mutations was higher in a subset of 178 cases that reported four Jewish grandparents (16.9%) than in cases who did not report Jewish ancestry (8.0%) (p < 0.01). Nine different GBA mutations were identified in PD cases, including 84insGG, E326K, T369M, N370S, D409H, R496H, L444P, RecNciI, and a novel mutation, P175P. CONCLUSIONS: This study suggests that the Glucocerebrosidase gene may be a susceptibility gene for Parkinson disease and that Glucocerebrosidase mutations may modify age at onset.

Biomarkers of oxidative stress in schizophrenic and control subjects.

Increasing evidence indicates that oxidative injury exists in schizophrenia. Although it may not be the main cause, oxidative damage has been suggested to contribute to the pathophysiology and may account for deteriorating course and poor outcome in schizophrenia. A human study was undertaken, therefore, to investigate possible differences in biomarkers of DNA, lipid and protein oxidation in schizophrenic (n=16) and control subjects (n=17). Plasma vitamin C levels were also compared in both groups. Cellular DNA damage and plasma protein carbonyl levels were increased in the schizophrenic group compared to control subjects but not significantly. However, DNA damage in lymphocytes from the male schizophrenic group was significantly higher than the female group. Biomarkers of lipid peroxidation and plasma vitamin C levels also revealed no significant difference between the two groups under investigation, although a significant elevation in plasma vitamin C was observed in the female control group when compared to the male groups.

Chronic cocaine administration reduces phospholipase A(2) activity in rat brain striatum.

BACKGROUND: Phospholipase A(2) (PLA(2)) catalyses the release of free fatty acids used for eicosanoid biosynthesis. We previously reported that calcium-stimulated PLA(2) activity is reduced in the brain of cocaine users and patients with schizophrenia, and have speculated that this is due to dopaminergic hyperactivity in both conditions. METHODS: To investigate these observations under controlled conditions, PLA(2) activity was measured in brain of rats exposed to cocaine and the dopamine receptor antagonist haloperidol. RESULTS: As compared with saline-treated controls, calcium-stimulated PLA(2) activity was reduced (-30%; P<0.01) in the dopamine-rich striatum of animals sacrificed 1 h after chronic (20 mg/kg/day) injection of cocaine, but was normal in haloperidol- (2 mg/kg/day) treated animals, and in the dopamine-poor cortex and cerebellum of animals treated with either drug. CONCLUSION: This confirms and extends our observations in human brain, and further suggests a link between the brain dopaminergic and phospholipid catabolic systems.

An animal model of nicotinic-acid-induced vasodilation: effect of haloperidol, caffeine and nicotine upon nicotinic acid response.

BACKGROUND: The normal vasodilatory response to ingestion of nicotinic acid (NA) is impaired in some patients with schizophrenia. It is unclear whether the impairment is a feature of the disorder itself or to a confounding factor such as neuroleptics, caffeine or nicotine use. METHODS: To address this question in a controlled manner, we have developed an animal (rat) model of NA-induced vasodilation, in which response is monitored by measuring change in skin temperature. RESULTS: We observed that (i) acute administration of acetylsalicylic acid (100mg/kg), caffeine (2.5mg/kg) and haloperidol (0.1 or 0.5mg/kg) and (ii) chronic administration of haloperidol (0.2mg/kg/day) significantly inhibited NA (30 mg/kg) response, whereas neither acute (0.25mg/kg) or chronic (0.5mg/kg/day for 14 days) administration of nicotine, or chronic administration of caffeine (5mg/kg/day for 14 days) had any significant effect upon NA response. CONCLUSIONS: Our data suggest that at least one drug commonly used to treat schizophrenia (haloperidol) can interfere with the vasodilatory response to NA. Studies using non-medicated patients with schizophrenia are required to determine whether reduced vasodilatory response to NA in schizophrenia is a feature of the disorder or a consequence of treatment.

Dopamine D1-stimulated adenylyl cyclase activity in cerebral cortex of autopsied human brain.

Although the cerebral cortical dopamine D(1) receptor is considered to play a role in normal and abnormal brain function, little information is available on its characteristics in human brain. We compared dopamine-stimulated adenylyl cyclase (AC) activity in homogenates of cerebral cortex (frontal, temporal, parietal, occipital and cingulate cortex) of autopsied brain of neurologically normal subjects to that in striatum. Cerebral cortical AC activity was modestly and dose-dependently stimulated by dopamine (maximal 20-30%) with low microM EC50s and such stimulation was inhibited by the selective dopamine D1 receptor antagonist SCH23390. The magnitude of the maximal stimulation by dopamine was similar in autopsied and biopsied cerebral cortex. The extent of maximal stimulation was similar to that in dopamine-rich striatum (caudate, putamen and nucleus accumbens), despite much lower density of dopamine D1 receptors in cerebral cortex vs. striatum. The EC50 for dopamine stimulation in cerebral cortex (approximately 1 microM) was lower than that for caudate and putamen (approximately 3 microM). No detectable dopamine stimulation was observed in cerebellar cortex, thalamus or hippocampus. Dopamine stimulation in both cerebral cortex and striatum was independent of calcium activation. We conclude that dopamine stimulated AC can be measured in cerebral cortex of human brain allowing for the possibility that this process can be examined in human brain disorders in which dopaminergic abnormalities are suspected.

Elevated activity of phospholipid biosynthetic enzymes in substantia nigra of patients with Parkinson's disease.

We reported that the activities of phospholipase A2, phosphocholine cytidylyltransferase and phosphoethanolamine cytidylyltransferase, key phospholipid metabolic enzymes, are low in substantia nigra of normal human brain and that this might reduce the ability of nigral neurons to repair damage to cell membranes. To determine whether adaptive changes in nigral phospholipid metabolism can occur in idiopathic Parkinson's disease we compared activities of 11 catabolic and anabolic enzymes in autopsied brain of 10 patients with Parkinson's disease to those in control subjects. Nigral activity of the catabolic enzyme phospholipase A2 was normal in the Parkinson's disease group, whereas that of the biosynthetic enzymes phosphoethanolamine cytidylyltransferase, phosphocholine cytidylyltransferase, and phosphatidylserine synthase were elevated 193, 48 and 38%, respectively, possibly representing a compensatory response to repair membrane phospholipids. Enzyme activities were normal in all other brain areas with the exception of increased (+26%) activity of calcium-stimulated phospholipase A2 in putamen, a change which could be consequent to either decreased dopaminergic striatal input or to a dopamine nerve terminal degenerative process. Our data indicate that the normally low rate of membrane phospholipid synthesis in the substantia nigra, the primary area of neurodegeneration in Parkinson's disease, is increased during the course of the disorder. We suggest that pharmacotherapies which augment this compensatory response might have utility as a treatment for Parkinson's disease.

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin.

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.

Abnormal activity of membrane phospholipid synthetic enzymes in the brain of patients with Friedreich's ataxia and spinocerebellar atrophy type-1.

Much evidence, derived from biochemical studies of both blood and autopsied brain, has suggested that phospholipid metabolism is abnormal in patients with Friedreich's ataxia (FA), a disorder characterized by severe neuronal loss in the spinal cord and lower brain stem with no, or only modest, damage in other brain regions. To establish the cause of our recent finding of reduced brain levels of phospholipids in FA, we assayed activities of 10 phospholipid-metabolizing enzymes in the autopsied cerebellar cortex of patients with the disorder and, for comparison, in a group of patients with spinocerebellar ataxia type 1 (SCA-1), a disease characterized, unlike FA, by marked neuronal loss in the cerebellar cortex. Enzyme activities were also measured in four brain areas which are relatively unaffected morphologically in both FA and SCA-1. We found that ethanolamine kinase activity was increased in multiple brain regions of patients with FA (increased 31%-137%) and, more modestly, in SCA-1 (increased 39%-60%), suggesting a nonspecific enhancement of phosphoethanolamine production in both disorders. In contrast, the activity of phosphoethanolamine cytidylyltransferase (PECT), the rate-limiting enzyme of phosphatidylethanolamine synthesis, was significantly and markedly decreased by 35%-78% in the cerebellar, frontal, and occipital cortices of patients with FA but was normal in SCA-1. Reduced PECT activity in FA may explain the lower brain levels of phosphatidylethanolamine in the disorder. Moreover, because decreased PECT activity in FA occurs in brain regions having no, or only modest, morphologic damage, this may represent a systemic change consequent to the frataxin gene defect. Our data also suggest that therapeutic intervention in FA designed to increase synthesis of membrane phospholipids may warrant further investigation.

Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.

Clinical subtyping reveals significant differences in calcium-dependent phospholipase A2 activity in schizophrenia.

BACKGROUND: Inconsistent results in the study of phospholipid metabolism in schizophrenia may reflect the heterogeneous nature of the illness(es). Differences in patients' responses to niacin, a compound causing vasodilation via stimulation of phospholipid dependent signaling cascades, defines more homogeneous patient subgroups in which the rate limiting enzyme of this signaling pathway, phospholipase A2 (PLA2), can be studied. METHODS: Subjects were categorized as niacin-insensitive (10 schizophrenic patients and 1 control) or niacin-sensitive (13 schizophrenic patients and 29 controls). Comparisons of serum calcium-dependent PLA2 were undertaken with and without consideration of niacin sensitivity. RESULTS: Significantly more schizophrenic patients were niacin-insensitive than controls (chi 2 (1) = 12.8, p < .001). Comparison of mean serum calcium-dependent PLA2 level of all schizophrenic subjects with all healthy controls revealed no statistical difference (t(51) = .79, NS). Subtyping the schizophrenia group by niacin sensitivity/insensitivity, however, allowed significant differences to emerge (F(2,49) = 4.40, p = .018). Post-hoc tests showed the mean PLA2 activity level of niacin-sensitive subjects was lower than that of healthy subjects. CONCLUSIONS: Treatment strategies which increase calcium-dependent PLA2 activity may aid in reducing states of excess dopaminergic activity by activating second messenger systems rather than receptor blockade.

Phosphatidylcholine and phosphatidylethanolamine metabolites may regulate brain phospholipid catabolism via inhibition of lysophospholipase activity.

Brain levels of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), abundant metabolites of phosphatidylcholine and phosphatidylethanolamine, are increased in several disorders of the human brain. To determine whether accumulation of these compounds may alter phospholipid metabolism, we assessed the ability of GPE and GPC to modulate the activities of phospholipase A(2), lysophospholipase, and other enzymes involved in phospholipid metabolism, in preparations of human brain parietal cortex. GPC and GPE acted as competitive inhibitors of lysophospholipase activity, but failed to alter the activity of the other enzymes tested. Our results suggest that GPC and GPE may normally act to inhibit lysophospholipid hydrolysis, thereby reducing the rate of membrane phospholipid degradation.

Modest cholinergic deafferentation fails to alter hippocampal G-proteins.

The integrity of hippocampal G-protein mediated signalling following ibotenate induced lesion of the medial septum was examined. The lesion was confined histologically to the septum and induced a 23% reduction in hippocampal choline acetyltransferase (ChAT) activity and G-proteins levels and related enzyme activities were measured in the hippocampus following a 21 day survival period. The relative levels of five G-protein subunits (Gbeta, G(alpha)o, G(alpha)i1, G(alpha)i2, and G(alpha)s-L), basal GTPase, the degree of carbachol- or baclofen-stimulated GTPase activities, and the basal and fluoroaluminate-stimulated adenylate cyclase activities were apparently unaffected. To determine if our assay methodology was sensitive to changes in pre-synaptic signalling, we compared G-protein density in synaptosomes with total hippocampal homogenates. The concentration of G(alpha)q/11, G(alpha)i1, and G(alpha)i2. were significantly lower in synaptosomes, while G(alpha)o, was only marginally reduced. Thus, modest lesions of the medial-septal nucleus fail to alter G-protein signalling. However, our findings that G-protein density is lower in synaptosomal membranes than in total homogenates, indicates that the analysis of signalling events in synaptosomes following deafferentation could clarify adaptive changes which may occur at the presynaptic level.

Probing the interaction of T7 RNA polymerase with promoter.

Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellent candidate for studying structural aspects of transcription, because unlike the eucaryotic and bacterial RNA polymerases, it is a single subunit enzyme and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying transcription using this enzyme is that the high-resolution crystal structure of T7 RNA polymerase has been solved. However, a cocrystal structure of the polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promoter with T7 RNA polymerase. We constructed promoters that were substituted with the photo-cross-linkable nucleotide 5-iodo uracil at every dT in the promoter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In the melting domain, the strongest contacts occurred at -3 and at -1 on the template strand while very weak cross-linking was seen at -2 and at -4 on the nontemplate strand. In the binding domain, the strongest contacts were seen at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-linking was poor in the intervening region between the binding and the melting domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding. For a systematic study designed to probe the nature of base-specific interactions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate > citrate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contacts, we observed that at -2, -4, and -17 the contacts had a hydrophobic component, while at many other positions there was no significant effect, suggesting that the contacts in the promoter-polymerase complexes were predominantly ionic but at certain positions nonionic interactions also existed. To localize a specific interaction in the melting domain, we proteolyzed the cross-linked T7 RNAP and analyzed the fragments using gel electrophoresis, mass spectrometry, and amino acid composition. High-resolution mapping indicated that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.

Differential alteration of phospholipase A2 activities in brain of patients with schizophrenia.

We recently reported that the activity of a calcium-independent subtype of phospholipase A2 is increased in blood of patients with schizophrenia. The present investigation examined whether similar changes take place in brain of patients with this disorder, and for comparison, in patients with bipolar disorder. The activity of two classes of PLA2, calcium-stimulated and independent, were assayed in autopsied temporal, prefrontal and occipital cortices, putamen, hippocampus and thalamus of 10 patients with schizophrenia, 8 patients with bipolar disorder and 12 matched control subjects. Calcium-independent PLA2 activity was increased by 45% in the temporal cortex of patients with schizophrenia as compared with the controls but was not significantly altered in other brain areas. In contrast, calcium-stimulated PLA2 activity was decreased by 27-42% in the temporal and prefrontal cortices and putamen, with no significant alterations in other brain regions. Brain PLA2 activity was normal in patients with bipolar disorder. Calcium-stimulated PLA2 activity was normal in cortex, cerebellum and striatum of rats treated acutely or chronically with haloperidol, whereas calcium-independent PLA2 activity was decreased in striatum of chronically treated animals, indicating that altered PLA2 activity in patients with schizophrenia is unlikely to be a direct effect of medication. Studies of the cellular role played by PLA2 suggest that decreased calcium-stimulated PLA2 activity, as also occurs in striatum of chronic human cocaine users, may be due, in part, to increased dopaminergic activity in the disorder, whereas increased calcium-independent PLA2 activity may be related to abnormal fatty acid metabolism and oxidative stress in schizophrenia.

Brain glyceraldehyde-3-phosphate dehydrogenase activity in human trinucleotide repeat disorders.

BACKGROUND: Although the abnormal gene products responsible for several hereditary neurodegenerative disorders caused by repeat CAG trinucleotides have been identified, the mechanism by which the proteins containing the expanded polyglutamine domains cause cell death is unknown. The observation that several of the mutant proteins interact in vitro with the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) suggests that interaction between the different gene products and GAPDH might damage brain neurons. OBJECTIVE: To measure the activity of GAPDH in postmortem brain of patients with CAG repeat disorders. PATIENTS AND METHODS: Activity of GAPDH was measured in morphologically affected and unaffected brain areas of patients with 4 different CAG repeat disorders (Huntington disease, spinocerebellar ataxia 1 [SCA1], SCA2, and SCA3-Machado-Joseph disease), in brains of patients with Friedreich ataxia (a GAA repeat disorder) and Alzheimer disease, and in brains of matched control subjects. RESULTS: Brain GAPDH activity was normal in all groups with the exception of a slight but statistically significant region-specific reduction in the patients with Huntington disease (caudate nucleus, -12%) and Alzheimer disease (temporal cortex, -19%). CONCLUSION: The presence of the polyglutamine-containing proteins in CAG repeat disorders does not result in substantial irreversible inactivation or in increased activity of GAPDH in human brain.

Brain phospholipids and fatty acids in Friedreich's ataxia and spinocerebellar atrophy type-1.

Previous studies of patients with spinocerebellar atrophy type 1 (SCA-1) and Friedreich's ataxia (FA) have suggested the occurrence of membrane disturbances in both disorders. We measured concentrations of phosphatidylcholine (PC), diacyl and plasmalogen phosphatidylethanolamine (PE), and phosphatidylserine (PS), along with their fatty acid profiles, in the brains of eight patients with Friedreich's ataxia (FA) and nine patients with dominantly inherited spinocerebellar atrophy type 1 (SCA-1). Compared with the controls, levels of all phospholipid types (PE, PS, and PC) were reduced in the cerebellar but not occipital cortex of SCA-1 patients. In contrast, in the FA group, levels of PS and PE, but not PC, were reduced in both cerebellar and occipital cortices. The fatty acid composition of individual brain phospholipids was altered in both FA and SCA-1 patients, most markedly in the plasmalogen PE and PS classes of cerebellar phospholipids. Given the neuropathologic characteristics of each disorder, it is likely that altered fatty acid composition and phospholipid levels in SCA-1 cerebellar cortex occur as a consequence of pronounced cerebellar degeneration. In contrast, reduced phospholipid levels in FA cerebellar and occipital cortex, areas characterized by, at most, minimal neuronal loss in FA, may represent a widespread alteration in cellular phospholipid metabolism occurring in response to the specific gene defect in the disorder.

RNA-binding site in T7 RNA polymerase.

Recent models of RNA polymerase transcription complexes have invoked the idea that enzyme-nascent RNA contacts contribute to the stability of the complexes. Although much progress on this topic has been made with the multisubunit Escherichia coli RNA polymerase, there is a paucity of information regarding the structure of single-subunit phage RNA polymerase transcription complexes. Here, we photo-cross-linked the RNA in a T7 RNA polymerase transcription complex and mapped a major contact site between amino acid residues 144 and 168 and probably a minor contact between residues 1 and 93. These regions of the polymerase are proposed to interact with the emerging RNA during transcription because the 5' end of the RNA was cross-linked. The contacts are both ionic and nonionic (hydrophobic). The specific inhibitor of T7 transcription, T7 lysozyme, does not compete with T7 RNA polymerase for RNA cross-linking, implying that the RNA does not bind the lysozyme. However, lysozyme may act indirectly via a conformational change in the polymerase. In the current model, the DNA template lies in the polymerase cleft and the fingers subdomain may contact or maintain a template bubble, and a region in the N terminus forms a partly solvent-accessible binding channel for the emerging RNA.

Mechanisms for the processing of a frozen topoisomerase-DNA conjugate by human cell-free extracts.

The metabolic fate of covalently linked DNA-protein complexes (cross-links) is not clearly understood. Our aim was to investigate the processing of protein-DNA cross-links by cellular enzymes. As an example of a DNA-protein cross-link, we have constructed frozen topoisomerase-DNA conjugates and investigated their processing by human cell-free extracts. A suicide DNA substrate was constructed that upon reaction with vaccinia type I topoisomerase yielded a highly stable covalent DNA-protein cross-link. When this conjugate was treated with human nuclear or whole cell extracts, two sites of DNA breakpoints were detected: one set of double-stranded breaks occurred close to the 3' side of the topoisomerase (topo) conjugation site, and there was another set of nicks about 30 nucleotides 3' to the topo site. The double-stranded breaks were not made by extracts from xeroderma pigmentosum group A mutant cells, suggesting that the xeroderma pigmentosum group A damage recognition protein may be required for the occurrence of DNA breakage. In addition to these DNA breakage reactions, there was an activity that resulted in the delinking of the frozen topoisomerase (or proteolytic fragments thereof) from the DNA substrate, which was followed by a ligation step that restored the continuity of the broken DNA strand at the erstwhile topo attachment site. We suggest that frozen topoisomerase-DNA conjugates (and perhaps other types of covalent DNA-protein complexes) are processed by multiple pathways that may involve the cleavage of the DNA in the covalent protein-DNA complex and/or enzymatic delinking followed by ligation of the broken DNA ends. These processes may represent the "repair" of DNA-protein cross-links.