Background and Objectives:
India is among the largest countries to implement the revised National Tuberculosis Control Program (RNTCP). This program provides intermittent regimens to the patients, where the doses of isoniazid and ethambutol are more as compared to the daily regimen, which is a cause of concern, particularly with regard to the ocular toxicity of ethambutol. The present study was undertaken to explore the ocular toxicity in the patients registered under the program.
Materials and Methods:
This was a prospective single center cohort study of 64 patients of categories I and II, coming to the RNTCP-Directly Observed Treatment Strategy (DOTS) center at a tertiary care referral hospital. The detailed history, best corrected visual acuity, fundus examination, and color vision test were carried out in all patients at the start of treatment and then at the first and second month of treatment.
Loss in visual acuity from the baseline was noted at the second month follow up in 12 (9.4%) eyes (P = 0.001), visual field defects were seen in eight (6.3%) eyes (P = 0.0412), and optic disc abnormalities were observed in six (4.7%) (P = 0.013) eyes. Color vision abnormalities were noted in 16 (12.6%) eyes (P = 0.003), four eyes showed impairment in red–green color perception, and the others showed impairment in blue–yellow color perception as well. Patients with ocular symptoms were advised to stop ethambutol and they showed improvement in visual acuity after follow up of one to two months. The overall outcome of treatment was not affected by discontinuation of ethambutol in these patients.
Ethambutol when taken according to program could cause ocular toxicity. The early recognition of ocular symptoms is important to prevent unnecessary delay in diagnosis and probable irreversible visual loss.
Ethambutol toxicity; ocular symptoms; revised national tuberculosis control program; visual defects
A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of Candida albicans, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of C. albicans. The lipid profiles of the YNB- and YPD-grown C. albicans cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the Candida cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of C. albicans. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of C. albicans, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.
Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C120-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1−/− mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in 1) controlling the PAR level after MMS exposure and 2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.
Obstructive sleep apnoea (OSA) and obstructive sleep apnoea syndrome (OSAS) are subsets of sleep-disordered breathing. Awareness about OSA and its consequences amongst the general public as well as the majority of primary care physcians across India is poor. This necessiated the development of the INdian initiative on Obstructive sleep apnoea (INOSA) guidelines under the auspices of Department of Health Research, Ministry of Health & Family Welfare, Government of India. OSA is the occurrence of an average five or more episodes of obstructive respiratory events per hour of sleep with either sleep related symptoms or co-morbidities or ≥ 15 such episodes without any sleep related symptoms or co-morbidities. OSAS is defined as OSA associated with daytime symptoms, most often excessive sleepiness. Patients undergoing routine health check-up with snoring, daytime sleepiness, obesity, hypertension, motor vehicular accidents and high risk cases should undergo a comprehensive sleep evaluation. Medical examiners evaluating drivers, air pilots, railway drivers and heavy machinery workers should be educated about OSA and should comprehensively evaluate applicants for OSA. Those suspected to have OSA on comprehensive sleep evaluation should be referred for a sleep study. Supervised overnight polysomnography (PSG) is the “gold standard” for evaluation of OSA. Positive airway pressure (PAP) therapy is the mainstay of treatment of OSA. Oral appliances are indicated for use in patients with mild to moderate OSA who prefer oral appliances to PAP, or who do not respond to PAP or who fail treatment attempts with PAP or behavioural measures. Surgical treatment is recommended in patients who have failed or are intolerant to PAP therapy.
Bariatric surgery; CPAP; Indian guidelines; OSA; OSAS; polysomnography; sleep apnoea; sleep study; Syndrome Z
It is now well-known that the enhanced expression of ATP binding cassette (ABC) and major facilitator superfamily (MFS) proteins contribute to the development of tolerance to antifungals in yeasts. For example, the azole resistant clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of Cdr1p and/or CaMdr1p belonging to ABC and MFS superfamilies, respectively. Hence, azole resistant isolates display reduced accumulation of therapeutic drug due to its rapid extrusion and that facilitates its survival. Considering the importance of major antifungal transporters, the focus of recent research has been to understand the structure and function of these proteins to design inhibitors/modulators to block the pump protein activity so that the drug already in use could again sensitize resistant yeast cells. The review focuses on the structure and function of ABC and MFS transporters of Candida to highlight the recent advancement in the field.
multidrug resistance; ABC transporters; MFS transporters; azoles; efflux pumps; Candida
Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.
Curcumin (CUR) shows antifungal activity against a range of pathogenic fungi, including Candida albicans. The reported mechanisms of action of CUR include reactive oxygen species (ROS) generation, defects in the ergosterol biosynthesis pathway, decrease in hyphal development, and modulation of multidrug efflux pumps. Reportedly, each of these pathways is independently linked to the cell wall machinery in C. albicans, but surprisingly, CUR has not been previously implicated in cell wall damage. In the present study, we performed transcriptional profiling to identify the yet-unidentified targets of CUR in C. albicans. We found that, among 348 CUR-affected genes, 51 were upregulated and 297 were downregulated. Interestingly, most of the cell wall integrity pathway genes were downregulated. The possibility of the cell wall playing a critical role in the mechanism of CUR required further validation; therefore, we performed specific experiments to establish if there was any link between the two. The fractional inhibitory concentration index values of 0.24 to 0.37 show that CUR interacts synergistically with cell wall-perturbing (CWP) agents (caspofungin, calcofluor white, Congo red, and SDS). Furthermore, we could observe cell wall damage and membrane permeabilization by CUR alone, as well as synergistically with CWP agents. We also found hypersusceptibility in calcineurin and mitogen-activated protein (MAP) kinase pathway mutants against CUR, which confirmed that CUR also targets cell wall biosynthesis in C. albicans. Together, these data provide strong evidence that CUR disrupts cell wall integrity in C. albicans. This new information on the mechanistic action of CUR could be employed in improving treatment strategies and in combinatorial drug therapy.
Metallothioneins (MTs) are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1) the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2) apoptosis and (3) the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC). No information is available on the gene expression of MT2A isoform in different types and grades of RCC.
Materials and Methods:
In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR) analysis was done for the MT2A gene expression using β-actin as an internal control. All statistical calculations were performed using SPSS software.
The MT2A gene expression was found to be significantly increased (P < 0.01) in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05), while no change was observed in high-grade tumor (grade III and IV) in comparison to adjacent normal renal tissue.
The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.
Clear cell carcinoma; grade; metallothionein; renal cell carcinoma; Real time polymerase chain reaction
The evolution of disease or the progress of recovery of a patient is a complex process, which depends on many factors. A quantitative description of this process in real-time by a single, clinically measurable parameter (biomarker) would be helpful for early, informed and targeted treatment. Organ transplantation is an eminent case in which the evolution of the post-operative clinical condition is highly dependent on the individual case. The quality of management and monitoring of patients after kidney transplant often determines the long-term outcome of the graft. Using NMR spectra of blood samples, taken at different time points from just before to a week after surgery, we have shown that a biomarker can be found that quantitatively monitors the evolution of a clinical condition. We demonstrate that this is possible if the dynamics of the process is considered explicitly: the biomarker is defined and determined as an optimal reaction coordinate that provides a quantitatively accurate description of the stochastic recovery dynamics. The method, originally developed for the analysis of protein folding dynamics, is rigorous, robust and general, i.e., it can be applied in principle to analyze any type of biological dynamics. Such predictive biomarkers will promote improvement of long-term graft survival after renal transplantation, and have potentially unlimited applications as diagnostic tools.
The evolution of disease or the progress of recovery of a patient is usually monitored by collecting physical parameters, which may be simply the body temperature for a common cold or properties of tissue samples for e.g., cancer. Most often clinical decisions are taken based on the current value or because of a sizable change of a relevant parameter. As more advanced diagnostic tools become available, and huge numbers of parameters can be collected at short, frequent time intervals, two related questions arise. The first is, which of the parameters provides relevant information on the progress of disease or recovery as opposed to noise? Is there more information that can be obtained from the history of the evolution of such parameters? Here we propose a novel approach that leads, for the specific case of recovery from kidney transplant, to a positive answer.
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD+ and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.
We have shown earlier that fluconazole (FLC) stress induces global changes in the lipidome of Candida albicans in clinically adapted isolates. However, several laboratories have developed adapted in vitro FLC resistant strains of C. albicans to study azole resistance mechanisms. This study aimed to identify the lipid changes associated with FLC resistance in these in vitro adapted isolates. Using comparative lipidomics and principal component and discriminant analyses, we observed gradual changes in several lipid classes and molecular species upon FLC exposure of in vitro resistant C. albicans strains. Although the lipid imprint of FLC in vitro resistant isolates was very distinct from that of clinical isolates of C. albicans, the overall changes in lipid class compositions were similar in both cases. For example, an increased sterol content and depleted sphingolipid levels were the salient features of FLC resistance in both conditions. Taken together, it appears that the overall cellular lipid homeostasis is a critical factor in the observed FLC resistance and in handling FLC stress in both clinical and laboratory situations. The new observations reported herein have implications for more efficacious antifungal drug development as well as understanding host–infectious agent interactions in postgenomics microbiology practice.
One-third of the total human population is infected with the Mycobacterium tuberculosis. This bacterium causes illness in up to 9 million people annually and is responsible for three deaths every minute world-wide.
To determine the association of serum zinc level with vitamin A level in active pulmonary tuberculosis (TB) cases.
Materials and Methods:
It was a cross-sectional study of 208 active pulmonary TB patients aged 18-55 years. Blood samples were obtained from these patients to determine the serum zinc and serum retinol levels.
The mean age of the patients was 30.56 (±11.38) years ranging from 18 years to 55 years. More than half (54.3%) of the patients were males and 63% were married. Body mass index of the patients was 18.40 ± 3.10. The serum zinc and vitamin A levels among the patients were 9.60 (±0.86) μmol/l and 0.77 (±0.22) μmol/l respectively. However, haemoglobin, white blood cell, erythrocyte sedimentation rate, and serum albumin were 10.02 (±1.33) g/dl, 10076.01 (±1822.67) cell/mm3, 14.50 (±2.95) mm/h and 3.40 (±0.32) g/dl respectively. There was a strong correlation between serum zinc and vitamin A levels (r = 0.86, P < 0.01). Vitamin A levels were not significantly different among the different age groups; however, this was significantly (P = 0.001) higher in male (0.82 ± 0.23, 95% confidence interval [CI] =0.77-0.86) patients as compared to females (0.71 ± 0.20, 95% CI = 0.67-0.75).
Zinc deficiency may indirectly influence the metabolism of Vitamin A via reduction of the levels of circulating proteins.
Deficiency; tuberculosis; vitamin A; zinc
Visual disturbance as a presenting feature of pseudohypoparathyroidism (PHP) is uncommon. Although papilledema is commonly reported with hypoparathyroidism primary or secondary, but not reported commonly with PHP.
Description of the Case:
A 10-year-old male child presented to our outpatient service with the complaints of blurring of vision, diplopia, and associated headache. There was no history of seizure episode. Patient had rounded face with a short, stocky built. Shortening of the fourth metacarpal and fifth metatarsal was present. Pitted nails and bilateral cataract. Patient also had clinical signs and biochemical parameters of hypocalcemia, along with normal parathyroid hormone (PTH) levels. Consistent with pseudohypopathyroidism.
In cases of chronic papilledema, the assessment of the calcium serum level is a safe and simple method to exclude hypoparathyroidism or PHP.
Pseudohypoparathyroidism; papilledema; AHO Phenotype
Swyer–James–MacLeod syndrome is characterised by unilateral hyperlucency on chest radiograph with small or normal-sized lung on the affected side and compensatory hyperinflation of opposite lung. Hyperinflation of the affected lung is a very rarely reported entity. An adult female patient, who presented with exertional breathlessness and diagnosed to have hypoplastic left pulmonary artery with hyperlucent, hyperinflated and herniated left lung is described.
Congenital muscular torticollis results from shortening or excessive contraction of the sternocleidomastoid (SCM) muscle. The reported incidence varies between 0.4 and 1.9%. Various theories have been proposed, but its true aetiology remains obscure. The deformity is characterized by a practically painless, contracted cordlike SCM muscle, which pulls the head toward the side affected, narrows and draws the shoulder upward, forcing the chin in the opposite direction. Torticollis of congenital origin is a deformity not commonly met with in the practice of maxillofacial surgery. The rarity, lack of, or inadequacy of the previous treatment, together with the advanced age and marked deformity appear to warrant an investigation and report of the outcome. A case of congenital muscular torticollis is presented who reported at the age of 18 years and has been successfully treated by unipolar SCM release.
Quid chewing practice has been a part of our tradition since centuries with little known evidence of oral cancer. However, recent trends show a rise in occurrence of oral cancer often associated with tobacco and arecanut usage. Ascorbic acid is an important salivary antioxidant. Betel leaf which is used in quid is known to contain ascorbic acid.
The aim of our study was to assess the salivary levels of ascorbic acid in traditional quid chewers so as to determine whether the betel leaf has protective antioxidant action.
Materials and Methods:
Salivary ascorbic acid levels of 60 subjects were estimated using the Dinitrophenyl hydrazine method.
The results revealed that quid chewers who used betel leaf had higher salivary ascorbic acid content compared to nonbetel leaf quid chewers. This could possibly be due to the protective antioxidants in the betel leaf.
Antioxidant; ascorbic acid; betel quid; piper betel
The aim of our study was to evaluate the advantages and disadvantages of 3D plating system in the treatment of mandibular fractures.
Patients and Methods:
20 mandibular fractures in 18 patients at various anatomic locations and were treated by open reduction and internal fixation using 3D plates. All patients were followed at regular intervals of 4th, 8th and 12th weeks respectively. Patients were assessed post-operatively for lingual splay and occlusal stability. The incidence of neurosensory deficit, infection, masticatory difficulty, non-union, malunion was also assessed.
A significant reduction in lingual splay (72.2%) and occlusal stability (72.2%) was seen. The overall complication rate was (16.6%) which included two patients who developed post-operative paresthesia of lip, three patients had infection and two cases of masticatory difficulty which later subsided by higher antibiotics and 4 weeks of MMF. No evidence of non-union, malunion was noted.
A single 3D 2 mm miniplate with 2 mm × 8 mm screws is a reliable and an effective treatment modality for mandibular fracture.
3D plates; mandible fractures; miniplates; lingual splay
This study shows that antifungal curcumin (CUR), significantly depletes ergosterol levels in Candida albicans. CUR while displaying synergy with fluconazole (FLC) lowers ergosterol. However, CUR alone at its synergistic concentration (lower than MIC50), could not affect ergosterol contents. For deeper insight of CUR effects on lipids, we performed high throughput mass spectroscopy (MS) based lipid profiling of C. albicans cells. The lipidome analysis revealed that there were no major changes in PGLs composition following CUR treatment of Candida, however, significant differences in molecular species of PGLs were detected. Among major SPLs, CUR treatment resulted in the reduction of ceramide and accumulation of IPCs levels. The lipidome of CUR treated cells confirmed a dramatic drop in the ergosterol levels with a simultaneous accumulation of its biosynthetic precursors. This was further supported by the fact that the mutants defective in ergosterol biosynthesis (ERG2 and ERG11) and those lacking the transcription factor regulating ergosterol biosynthesis, UPC2, were highly susceptible to CUR. Our study first time shows that CUR, for its antifungal activity, targets and down regulates Δ5, 6 desaturase (ERG3) resulting in depletion of ergosterol. This results in parallel accumulation of ergosterol biosynthetic precursors, generation of ROS and cell death.
Candida albicans; Curcumin; Ergosterol; Δ5,6; desaturase Lipid homeostasis
Background & objectives:
ADAM33 is a member of a family of genes that encode membrane-anchored proteins with a disintegrin and a metalloprotease domain, primarily expressed in lung fibroblasts and bronchial smooth muscle cells. ADAM33 has been identified as a risk factor for asthma and is known as a gene associated with airway remodelling. The present study was conducted with the aims to investigate the expression of ADAM33 protein in patients of asthma and non-asthmatic controls, and to assess if the expression of ADAM33 protein relates with severity of asthma.
A total of 35 subjects, including 27 patients with asthma and eight non-asthmatic controls were included using Global Initiative for Asthma guidelines 2005. Bronchial biopsy tissues were collected and paraffin sections were made to store all study samples. Immunohistochemistry was performed using standardized protocol.
An increase in expression of ADAM33 protein was observed in the epithelium, smooth muscle and mesenchymal cells of asthma cases when compared to controls but there was no relationship with severity of asthma.
Interpretation & conclusions:
A higher expression of ADAM33 protein was seen in asthma patients compared to controls. Large prospective studies need to be done with adequate study design to confirm these preliminary finding.
ADAM33 protein expression; asthma; bronchial biopsy; immunohistochemistry; severity of asthma
Previous studies have reported that the dental follicular tissues associated with impacted lower third molars (ILTMs) may undergo cystic degeneration and/or neoplastic transformation. This is especially likely when the pericoronal space is >2.5 mm on intraoral radiographs and >3 mm on panoramic radiographs and to examine dental follicular tissue for pathological changes in patients with ILTMs and pericoronal radiolucencies of <2.5 mm.
Histopathological evaluation of follicular tissues associated with ILTMs.
Materials and Methods:
The morphology of the hematoxylin and eosin-stained follicular tissues of 146 such impactions were studied.
On microscopy, no cystic structures with fibrous walls were identified. 85 cases (58%) showed fibrous or myxomatous connective tissue and no epithelial elements. 61 cases (42%) showed epithelial elements in addition to fibrocollagenous tissue. Of these, 16 cases exhibited epithelium, of which 13 cases showed reduced enamel epithelium and three cases showed squamous metaplasia/non-keratinized stratified squamous epithelium.
All asymptomatic unerupted third molars with pericoronal radiolucency of <2.5 mm should be retained since they do not exhibit cyst formation microscopically.
Dental follicle; impacted lower third molar; pathology
Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.
To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in Saccharomyces cerevisiae, we replaced the RAD27FEN1 open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In S. cerevisiae, Rad27Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in S. cerevisiae, and this partially rescued the MMS hypersensitivity observed with rad27fen1-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in rad27fen1-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.
During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an ‘affinity-capture’ procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3′-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3′-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3′-blocked intermediate.