Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gyrate atrophy (GA) is an autosomal recessive chorioretinal degenerative disease of the eye caused by an inborn defect of the nuclear encoded mitochondrial enzyme ornithine aminotransferase (OAT). We have described previously a GA patient with a 5.0-kilobase pair truncated EcoRI OAT gene fragment and the absence of OAT mRNA on Northern blot analysis. Cloning and sequencing analysis of the truncated gene fragment revealed a 1,072-base pair (bp) deletion including the entire exon 6, starting in intron 5, 172 bp upstream of exon 6 and ending in intron 6, 772 bp downstream of exon 6. A short direct repeat sequence (AGGAGC), resembling the sequence shown to cause DNA polymerase alpha to pause, and sequences capable of forming hairpin loops were both present at the 5' and 3' break-points of the deletion. Reverse transcription-polymerase chain reaction amplification of the patient's RNA with OAT primers yielded DNA fragments of two different sizes, consistent with a low level expression of OAT mRNA. Direct sequencing of the smaller fragment demonstrated the complete absence of exon 6 sequence in the mRNA predicted from the deletion, causing a reading frame shift which results in a premature termination codon at position 192. The mutation in the other allele has been demonstrated by polymerase chain reaction, denaturing gradient gel electrophoresis, and direct sequencing also to be a premature termination codon in exon 6. The absence of detectable OAT mRNA in this patient is consistent with these premature termination mutations because they have been shown to decrease the level of mRNA, especially if present early in the coding sequence.
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PMID:A deletion in the ornithine aminotransferase gene in gyrate atrophy. 161 92

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.
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PMID:Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs. 1274 17