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Biomedical subjects

P Robins

Publications and source records attributed to P Robins.

At least 19 recordsLinked to original sources
PubMed2001

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.

DNA Damage↗
PubMed2001

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil-DNA glycosylase.

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.

Animals↗
PubMed2000

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

Animals↗
PubMed1999

Molecular characterization of a human DNA kinase.

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5'-hydroxyl termini and dephosphorylate 3'-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5'-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.

Amino Acid Sequence↗
PubMed1999

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.

Amino Acid Sequence↗
PubMed1999

Microcystic adnexal carcinoma: collaborative series review and update.

BACKGROUND: Microcystic adnexal carcinoma (MAC) is a malignant appendageal tumor first described in 1982. It can be clinically and histologically confused with other malignant and benign cutaneous neoplasms, leading to inadequate initial treatment. This neoplasm is locally aggressive and deeply infiltrating, characterized by high morbidity and frequent recurrence. Mohs micrographic surgery has been used to conserve tissue and improve the likelihood for cure. OBJECTIVE: We report our experience using Mohs micrographic surgery for the treatment of MAC and compare with earlier reports in the literature. In addition, we review the epidemiology, clinical and histologic characteristics, and optimal treatment of this rare neoplasm. We also describe a 15-year-old white male patient with MAC on the scalp occurring only 7 years after radiation exposure. METHODS: The medical records of 11 patients with MAC who were treated by Mohs micrographic surgery were reviewed at both departments, and follow-up data were obtained. RESULTS: In all patients treated with Mohs micrographic surgery, there were no recurrences after a mean follow-up of 5 years. CONCLUSION: Mohs technique enables the detection of clinically unrecognizable tumor spread and perineural invasion often encountered with MAC. Aggressive initial treatment by microscopically controlled excision appears to offer the greatest likelihood of cure for this neoplasm, while providing conservation of normal tissue. In addition, we describe the second youngest patient with MAC and readdress the issue of previous radiotherapy as an important predisposing factor.

Adolescent↗
PubMed1998

Thoracic chordoma mimicking a neurogenic tumour.

A case of chordoma arising in the thoracic spine of a 20-year-old man is presented. Plain film and magnetic resonance imaging findings are included. The case is unusual in that the neoplasm presented as dumb-bell-shaped extradural mass, traversing and expanding a thoracic neural foramen and thereby mimicking a neurogenic tumour.

Adult↗
PubMed1997

Activation of the transcription factor Gli1 and the Sonic hedgehog signalling pathway in skin tumours.

Sporadic basal cell carcinoma (BCC) is the most common type of malignant cancer in fair-skinned adults. Familial BCCs and a fraction of sporadic BCCs have lost the function of Patched (Ptc), a Sonic hedgehog (Shh) receptor that acts negatively on this signalling pathway. Overexpression of Shh can induce BCCs in mice. Here we show that ectopic expression of the zinc-finger transcription factor Gli1 in the embryonic frog epidermis results in the development of tumours that express endogenous Gli1. We also show that Shh and the Gli genes are normally expressed in hair follicles, and that human sporadic BCCs consistently express Gli1 but not Shh or Gli3. Because Gli1, but not Gli3, acts as a target and mediator of Shh signalling, our results suggest that expression of Gli1 in basal cells induces BCC formation. Moreover, loss of Ptc or overexpression of Shh cannot be the sole causes of Gli1 induction and sporadic BCC formation, as they do not occur consistently. Thus any mutations leading to the expression of Gli1 in basal cells are predicted to induce BCC formation.

Animals↗
PubMed1997

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Animals↗
PubMed1997

P53 oncoprotein expression and gene mutations in some keratoacanthomas.

OBJECTIVE: To analyze the relationship of p53 oncoprotein overexpression in most keratoacanthomas (KAs) with gene mutations. DESIGN: Expression of p53 oncoprotein in immunohistochemical staining and its correlation to gene mutations in DNA extracted from KAs and tested in single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SETTING: A micrographic surgery unit and a dermatopathology unit at a university medical center. PATIENTS: Sixteen formalin-fixed, paraffin-embedded skin biopsy specimens were retrieved from dermatopathology archives. Biopsies were performed to establish the diagnosis of KA before surgical treatment. MAIN OUTCOME MEASURES: Intensity of staining in immunohistochemical testing for p53 oncoprotein expression and sequencing of gene mutations. RESULTS: Immunohistochemical staining of 16 KA specimens detected p53 oncoprotein in 15 (94%), distributed as strong in 4 (25%), moderate in 2 (12%) mild in 9 (56%), and negative in 1 (6%), compared with control specimens. Specimens were screened by SSCP for mutations in the p53 gene, and 1 specimen showed a potential mutation in exon 7. Direct sequencing of the samples revealed 2 point mutations. One specimen showed a change of G:A for A:G in codon 146 of exon 5, predicting an amino acid substitution of tryptophan for a stop codon. Another specimen revealed a change of T:A for A:T in codon 234 of exon 7, predicting an amino acid substitution of tyrosine for asparagine. CONCLUSIONS: Ninety-four percent of KA specimens evaluated had detectable p53 oncoprotein. This protein was associated with a point mutation in the p53 gene in 2 of 16 KAs evaluated. In a small fraction of KAs, overexpression of p53 oncoprotein may be associated with point mutations in the p53 gene.

Aged↗