Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.
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PMID:Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse. 845 36

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts from such patients are extremely sensitive to mutations induced by UV radiation, and the spectrum of mutations induced in their hypoxanthine phosphoribosyltransferase (HPRT) gene differs significantly from that seen in normal cells. To determine if this UV hypermutability reflects abnormally slow excision repair of cyclobutane pyrimidine dimers (CPD) or 6-4 pyrimidine-pyrimidones (6-4s) in that gene, we synchronized XP variant and normal fibroblasts, irradiated them in early G1-phase, 12 or more hours prior to the scheduled onset of S phase, harvested them immediately or after allowing various times for repair, and analyzed the DNA for photoproducts in the HPRT gene, using quantitative Southern blotting. To incise the DNA at CPD, we used T4 endonuclease V; to incise at 6-4s, we first used photolyase and UV365nm to reverse CPD and then UvrABC excinuclease. Excision of CPD was rapid, preferential, and strand-specific, but there was no significant difference in rate between the two kinds of cells. The half life was 4 h in the transcribed strand of the gene and 6.5 h in the nontranscribed strand. For excision of CPD in the genome overall, this value is 12 h. Excision of 6-4s from either strand of the HPRT gene was extremely rapid and preferential in both kinds of cells, with a half life of approximately 30 min. The results indicate that the UV hypermutability of the XP variant cells cannot be caused by slower rates of repair of CPD and/or 6-4s in the target gene for mutagenesis.
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PMID:Comparison of the rate of excision of major UV photoproducts in the strands of the human HPRT gene of normal and xeroderma pigmentosum variant cells. 853 50

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.
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PMID:Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair. 866 88

Deficiency of the enzyme adenine phosphoribosyltransferase (APRT) has been associated with hypersensitivity to the mutagenic effects of ethyl methanesulphonate (EMS) and 254 nm ultraviolet (UV) radiation in clone 707 of Friend mouse erythroleukaemia (FEL) cells. The molecular nature of spontaneous EMS- and UV-induced mutations in the coding region of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was determined for wild-type FEL cells and two APRT-deficient mutant sub-clones which have significantly reduced ATP pool levels, and are mutagen-hypersensitive. Mis-sense base substitutions were the predominant type of spontaneous mutation. However, exon deletions, possibly involving aberrant splicing of HPRT mRNA, and a non-sense mutation were also observed. EMS-induced mutations in wild-type and APRT-deficient mutant sub-clones were GC-->AT transitions, which is consistent with O6-ethylguanine being the primary pre-mutagenic lesion. All UV-induced mutations in both cell types were targeted to dipyrimidine sites where the two most common classes of photoproducts (cyclobutane pyrimidine dimers and [6-4] photoproducts) are formed. The similarity in the mutations observed in both cell types indicates that the mutagen hypersensitivity of APRT-deficient cells may be the result of decreased efficiency in the excision repair processes due to reduced levels of ATP.
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PMID:Molecular mechanisms of mutagen hypersensitivity in adenine phosphoribosyl transferase-deficient Friend mouse erythroleukaemia cells. 949 94

We investigated the relationship between nucleotide excision repair (NER) activity and apoptosis in UV-irradiated cells. Mouse erythroleukemia (MEL) and lymphoma (GRSL) cells exhibited enhanced sensitivity to the cytotoxic effects of UV radiation compared to hamster cell lines, although normal UV-induced hprt mutation frequencies were found. Determination of UV-induced repair replication revealed a limited capacity of MEL and GRSL cells to perform NER consistent with poor removal of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts from transcriptionally active genes during the first 8 h after UV exposure. However, both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts appeared to be processed to almost normal level 24 h after UV treatment. In parallel, we observed that the UV-irradiated MEL and GRSL cells suffered from severe DNA fragmentation particularly 24 h after UV exposure. Taken together, these data indicate a reduced repair of UV-induced photolesions in apoptotic cells, already established at the early onset of apoptosis. To test whether inhibition of repair in cells was due to inactivation of NER or to apoptosis-induced chromatin degradation, we performed in vitro excision assays using extracts from UV-irradiated MEL cells. These experiments showed that the NER capacity during early apoptosis was intact, indicating that slow removal of UV-induced photolesions in apoptotic cells is due to substrate modification (presumably degradation of chromatin) rather than direct inhibition of factors involved in NER.
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PMID:Impairment of nucleotide excision repair by apoptosis in UV-irradiated mouse cells. 958 42

In previously reported studies, we transfected repair-proficient murine fibroblasts with the denV gene of bacteriophage T4 and showed that expression of encoded endonuclease V markedly enhanced cyclobutane pyrimidine dimer (CPD) repair and reduced the frequency of ultraviolet radiation (UV)-induced mutations. In the present studies, we compared the spectra of UV-induced mutations at the hprt locus in denV-transfected and control cells. A significant difference in mutation types was observed. While multiple base deletions and single base insertions were found in denV-transfected but not control cells, multiple tandem and non-tandem point mutations identified in control cells were absent in denV-transfected cells. When we compared colony survival following UV exposure in the two cell lines, it appeared that endonuclease V expression did not enhance UV resistance, instead denV-transfected cells had increased susceptibility to low fluences of UV. The effects of endonuclease V expression on UV resistance and on UV mutational spectrum are likely to be due both to the removal of CPDs and to the novel enzymatic activity of endonuclease V.
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PMID:Altered UV resistance and UV mutational spectrum in repair-proficient murine fibroblasts expressing endonuclease V. 963 44

We have determined the in vitro DNA damage distribution induced by 254 mm UV in the human hprt gene. The sequence-specific nature of the DNA damage for both main classes of UV-induced photoproducts, i.e., cyclobutane pyrimidine dimers (CPDs) and the pyrimidine <6-4> pyrimidone photoproducts (64PyPy), was evaluated. Utilizing an automated DNA sequencer plus auxiliary software, semi-automated analyses were performed for peak quantitation and retention-time to sequence-position correlation. 64PyPy were predominantly formed at 5'-YTC-3' sites (p < 0.02; where Y = C,T). The effect of the 3'flanking nucleotide on the 64PyPy formation at 5'-TC-3' sites was 64PyPy at 5'-TCT-3' sites were induced at lower frequencies compared to 5'-TCM-3' sites (where M = A or C; p < 0.03). No effect of flanking nucleotides was detected for CPDs recovered at 5'-TT-3' sites. Sites of mutations in the hprt gene were compared to the sites of DNA damage. Two regions of frequently mutated nucleotides corresponded to sites of high deposition of damage. The two sites either had a high frequency of CPDs or 64PyPy, which implicated both types of photoproducts as premutagenic lesions.
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PMID:Correlation of UV-induced mutational spectra and the in vitro damage distribution at the human hprt gene. 972 24

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.
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PMID:Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice. 1003 86

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.
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PMID:Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding. 1033 13

The human population is exposed to both the ultraviolet A (UVA) and B (UVB) regions of the solar spectrum. UVB induces mainly dipyrimidine photoproducts in DNA by a direct photochemical mechanism, whereas UVA is absorbed by other cellular constituents and induces mainly oxidative damage indirectly. The proportions of the different dipyrimidine photoproducts, and the ratio of dipyrimidine to oxidative damage depend on the exact spectral output of a UV source. Irradiation of human epidermal keratinocytes induces release of cytokines, with cyclobutane pyrimidine dimers playing a significant role in the process. These cytokines may then modulate the activity of cells of the immune system. Freshly isolated human lymphocytes are exquisitely sensitive to UVB irradiation, because of their low deoxyribonucleotide pools. They also have a separate defect in removal of cyclobutane pyrimidine dimers from their DNA. We have observed that frequencies of mutations at the hprt locus in human T-lymphocytes and translocations involving the bcl2 locus in B-lymphocytes appear to be associated with sunlight levels over the period before the blood sample was taken. This may be an indirect cytokine-mediated effect, and may be relevant to the possible link between non-Hodgkin's lymphoma and sunlight. On the other hand, sunlight can have beneficial effects, and may protect against autoimmune diseases including type I diabetes and multiple sclerosis.
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PMID:Possible effects of sunlight on human lymphocytes. 1070 50


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