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)

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.
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PMID:Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease. 302 89

The sequence selectivity of DNA alkylation by the recently isolated pluramycin antitumour antibiotic DC92-B has been investigated using two methods: a piperidine-induced strand-breaking procedure and a Taq DNA polymerase/linear amplification method. These techniques reveal that guanines are the most reactive sites for alkylation and that the level of adduct formation at these sites is clearly sequence dependent. The highest levels of alkylation occurred at isolated guanines located in 5'-CGT sequences and also at the 5'-G in some 5'-CGG sequences. Isolated guanines in 5'-TGT sequences were also quite reactive. We have also re-examined, in parallel, the sequence selectivity of binding of the structurally-related compound hedamycin: the first known example of a bis(epoxide)-containing, DNA-alkylating pluramycin. Our studies included a more extensive sequence analysis of hedamycin binding than that previously reported and we are able, therefore, to define more precisely the sequence preference. Despite significant differences in the stereochemistry and substitution of their bis(epoxide) sidechains, hedamycin and DC92-B exhibited very similar sequence selectivities in our assays.
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PMID:Comparison of the sequence selectivity of the DNA-alkylating pluramycin antitumour antibiotics DC92-B and hedamycin. 769 48

The sequence specificity of the pluramycin antibiotics hedamycin and DC92-B, was established in intact human cells using a linear amplification system. In this system an oligonucleotide primer is extended by Taq DNA polymerase up to a damage site. The products are run on a DNA sequencing gel and the damage can be determined to the exact base pair. The human repetitive alpha RI DNA was used as the target DNA sequence for these experiments. It was found that G residues were the main site of adduct formation, for both hedamycin and DC92-B. The sequences 5'-TGT and 5'-CGT were the most intense sites of DNA damage. A comparison of the DNA damage intensity in intact cells and purified DNA revealed that the sequence position of adduct formation was very similar in the two environments. However, a densitometric comparison of the damage intensity in the two environments revealed significant differences. Two regions were found (120 and 130 bp in length) where the damage intensity was relatively lower in intact cells compared to purified DNA. But at the boundaries of these sequences, there were regions (approx. 50-60 bp long) that were relatively more damaged in intact cells compared to purified DNA. One explanation of this phenomenon is the presence of a protecting nucleosome core on each of the 120/130 bp regions and flanking nucleosome linker regions of 50-60 bp. This postulated sequence phasing of the nucleosomes corresponds almost exactly with the major nucleosome phasing found in African green monkey cells. Also the centromere protein B binding site is found in the border region between the nucleosome core and linker DNA regions. Hedamycin and DC92-B produced nearly identical results in this human cell system.
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PMID:The interaction of hedamycin and DC92-B in a sequence selective manner with DNA in intact human cells. 771 Oct 62

The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.
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PMID:In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass. 1095 33

The identification of point mutations is particularly essential in the fields of medical diagnosis and prognosis of many pathogenic and genetic diseases. In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed. In the assay, an allele-discriminating padlock probe was designed for targeting the sequence in the p53 oncogene locus. A circular template generated by enzymatic ligation upon the recognition of a point mutation (CGT to CAT) on the oncogene could be amplified isothermally by Phi 29 DNA polymerase. The elongated products, containing hundreds of copies of the circular DNA template sequence, were hybridized with Ru(bpy)(3)(2+) (TBR)-tagged probes and then captured onto streptavidin-coated paramagnetic beads. The resulting products were analyzed by magnetic bead based ECL platform. As low as 2 amol of mutated strands was detected by this assay, which could be attributed to the high amplification efficiency of Phi 29 DNA polymerase and current magnetic bead based ECL detection platform. In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site. It is demonstrated that this proposed method provides a highly sensitive and specific approach for point mutation detection.
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PMID:Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation. 2003 81