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

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.
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PMID:Cloning, characterization, and properties of seven triplet repeat DNA sequences. 866 77

Several human hereditary neuromuscular and neurodegenerative diseases are caused by abnormal expansion of triplet repeat sequences (TRSs) CAG/CTG, CGG/CCG, or GAA/TTC on certain chromosomes. It is generally accepted that multiple slippage synthesis accounts for the instabilities of TRS. Earlier in vitro experiments by Behn-Krappa and Doerfler showed that TRS with high GC content can be expanded. In contrast, here we demonstrated that certain AT-rich TRSs, (TTC)17, (GAA)10/(TTC)10 and (GAA)17/(TTC)17, were also expansion-prone in PCR. With respect to the sequence of TRS, surprisingly, we found that the AT-rich (GAA)17/(TTC)17 extended more efficiently than the GC-rich (CAG)17/(CTG)17. This strongly suggested that the AT content of the repeat may influence TRS expansion. Furthermore, to examine the expansion of single-stranded TRS, we showed that only (TTC)17, but not the complementary (GAA)17, can be expanded. This suggested that a T-T mismatch may stabilize compatible secondary structures, most likely hairpins, for slippage synthesis. However, another poly-pyrimidine TRS, (CCT)17, is not amplification-prone in PCR. Due to the high C-content, this TRS is unlikely to adopt hairpin structures at the high pH used for PCR. Thus, the single-stranded PCR experiment may serve as an indirect assay for the ability of a sequence to adopt a hairpin conformation. When amplification was performed in reactions using Klenow DNA polymerase, only the double-stranded TRSs can be expanded. The reaction rate for (GAA)10/(TTC)10 was slower than for (GAA)17/(TTC)17, suggesting that the length of the repeat may be important for the amplification of TRS. The findings of these in vitro experiments may aid in understanding TRS expansion in vivo.
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PMID:Amplification of GAA/TTC triplet repeat in vitro: preferential expansion of (TTC)n strand. 968 24

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.
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PMID:Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification. 1557 69