Excision of formamidopyrimidine lesions by endonucleases III and VIII is not a major DNA repair pathway in Escherichia coli

Proper maintenance of the genome is of great importance. Consequently, damaged nucleotides are repaired through redundant pathways. We considered whether the genome is protected from formamidopyrimidine nucleosides (Fapy•dA, Fapy•dG) via a pathway distinct from the Escherichia coli guanine oxidation system. The formamidopyrimidines are produced in significant quantities in DNA as a result of oxidative stress and are efficiently excised by formamidopyrimidine DNA glycosylase. Previous reports suggest that the formamidopyrimidine nucleosides are substrates for endonucleases III and VIII, enzymes that are typically associated with pyrimidine lesion repair in E.coli. We investigated the possibility that Endo III and/or Endo VIII play a role in formamidopyrimidine nucleoside repair by examining Fapy•dA and Fapy•dG excision opposite all four native 2′-deoxyribonucleotides. Endo VIII excises both lesions more efficiently than does Endo III, but the enzymes exhibit similar selectivity with respect to their action on duplexes containing the formamidopyrimidines opposite native deoxyribonucleotides. Fapy•dA is removed more rapidly than Fapy•dG, and duplexes containing purine nucleotides opposite the lesions are superior substrates compared with those containing formamidopyrimidine–pyrimidine base pairs. This dependence upon opposing nucleotide indicates that Endo III and Endo VIII do not serve as back up enzymes to formamidopyrimidine DNA glycosylase in the repair of formamidopyrimidines. When considered in conjunction with cellular studies [J. O. Blaisdell, Z. Hatahet and S. S. Wallace (1999) J. Bacteriol., 181, 6396–6402], these results also suggest that Endo III and Endo VIII do not protect E.coli against possible mutations attributable to formamidopyrimidine lesions

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Ferrocenylnaphthalene Diimide-Based Electrochemical Detection of Aberrant Methylation in hTERT Gene

Since aberrant methylation at CpG sites is linked to the silencing of tumor suppressor genes, DNA methylation analysis is important for cancer diagnosis. We developed ferrocenylnaphthalene diimide (FND), which has two ferrocenyl moieties at the substituent termini, as an electrochemical indicator for hybridized DNA duplexes. In this study, we attempted to detect aberrant methylation of human telomerase reverse transcriptase gene (hTERT), an efficient cancer marker, using FND-based hybridization coupled with electrochemical detection via a multi-electrode chip

Fluorescence-guided bone resection by using Visually Enhanced Lesion Scope in diffuse chronic sclerosingosteomyelitis of the mandible: clinical and pathological evaluation

Diffuse chronic sclerosingosteomyelitis (DCSO) is a refractory disease, becausethe etiology and pathogenesis remain poorly understood and to determine the border betweenunhealthy boneandhealthybone is difficult. However, progressive inflammation, clinical symptoms and a high recurrence rate of DCSO were the reasons for surgical treatment. We report a case of a 66-year old woman with DCSO of the right side of mandible who was treated with hemimandibulectomy and simultaneous reconstruction by vascularized free fibula flap. After preoperative administration of minocycline for 1 month, the bone fluorescence was successfully monitored by using a Visually Enhanced Lesion Scope (VELscope®). Intraoperatively, we could determine the resection boundaries. We investigated the clinical and histopathological findings. The fluorescence findings were well correlated with histopathological findings. Using a VELscope®was handy and useful to determine the border between DCSO lesion andhealthybone. The free fibula flap under the minocycline-derived bone fluorescence by using a VELscope®offered a good quality of mandibular bone and the successful management of an advanced and refractory DCSO

Screening for Oral Cancer Using Electrochemical Telomerase Assay

Electrochemical telomerase assay (ECTA) developed by our group was evaluated in an oral cancer screening using exfoliated oral cells and tissues obtained from patients of oral cancer, mucosa associated disease, or healthy volunteers. Telomerase activity from ECTA is correlated with hTERT mRNA expression level using a real‐time PCR and was increasing in the following order: healthy volunteer group<mucosa associated disease group<oral cancer group. Sensitivity and specificity of ECTA were 88% and 72%, respectively when used 17% of the threshold value based on the receiver operating characteristic curve in ECTA data

Electrochemical telomerase assay for screening for oral cancer

Telomerase has long been known to be a marker for cancer. We have developed a new method of detecting it: the electrochemical telomerase assay (ECTA). We have previously confirmed that the assay is easier to do and more precise than the conventional telomeric repeat amplification protocol, which is currently the most widely used. Here we describe a pilot study made to establish a screening system for oral cancer using ECTA. We evaluated three types of clinical samples obtained from 44 patients with oral cancer and 26 healthy volunteers: exfoliated cells from the whole oral cavity, exfoliated cells from local lesions, and tissue from the lesion itself. The current increase ratio (Δi) obtained by ECTA was significantly higher in the oral cancer group for each type of sampling used. The threshold value for Δi was 19% when calculated by analysis of receiver-operating characteristic curves. Sensitivity and specificity values were 86% and 85% for cells from the oral cavity, 82% and 85% in cells from local lesions, and 95% and 92% in cells from the tumour itself, respectively. There were also no significant differences in sensitivity and specificity associated with age, size of tumour, site of lesion, or degree of malignancy. ECTA therefore seems to be a promising assay for screening for oral cancer

Oral Cancer Screening Based on Methylation Frequency Detection in hTERT Gene Using Electrochemical Hybridization Assay via a Multi‐electrode Chip Coupled with Ferrocenylnaphthalene Diimide

Ferrocenylnaphthalene diimide‐based electrochemical hybridization assay via a multi‐electrode chip was applied to detect the methylation frequency in the promoter region of human telomerase reverse transcriptase (hTERT) gene for clinical samples from tissues, local exfoliated oral cells from a lesion, or from entire oral cavity after their methylation specific PCRs. These methylation frequencies were increased with cancer progress as the following order: healthy volunteers, oral leukoplakia as precancerous lesion, and oral squamous cell carcinoma (OSCC). Operating characteristic analysis of the obtained current data doesn\u27t only give excellent discrimination ability of OSCC, but also of oral leukoplakia from healthy volunteers for all samples. Sensitivity and specificity was 95% and 90%, respectively, which is a comparable with methods in practical use