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)

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

In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.
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PMID:Detection of K-ras point mutation by in situ PCR in cell suspensions: comparison of the indirect and direct methods. 923 54

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC --> TA, GC --> AT, etc.) principally via its major adduct [+ta]-B[a]P-N2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P-N2-dG built into a plasmid in a 5'-TGT sequence gives approximately equal numbers of G --> T and G --> A mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in G --> T versus G --> A mutations. G --> T mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V ((delta)umuD/C), demonstrating a role for damage-inducible DNA Pol V in a G --> T pathway. G --> T mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5'-GGA sequence, and showed that the frequency of G --> T mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two G --> T mutagenic pathways exist for [+ta]-B[a]P-N2-dG, where sequence context dictates which pathway is followed. In contrast to the G --> T mutations, herein G --> A mutations from [+ta]-B[a]P-N2-dG in the 5'-TGT sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that G --> A mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P-N2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two G --> A mutagenic pathways for [+ta]-B[a]P-N2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both G --> T and G --> A mutations from [+ta]-B[a]P-N2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.
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PMID:A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli. 1517 47

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is activated to (+)-anti-B[a]PDE, which induces a variety of mutations (e.g., G --> T, G --> A, etc.) via its major adduct [+ta]-B[a]P-N2-dG. One hypothesis is that adducts (such as [+ta]-B[a]P-N2-dG) induce different mutations via different conformations, probably when replicated by different lesion-bypass DNA polymerases (DNAPs). We showed that Escherichia coli DNAP V was responsible for G --> T mutations with [+ta]-B[a]P-N2-dG in a 5'-TGT sequence (Yin et al., (2004) DNA Repair 3, 323), so we wish to study conformations of this adduct/sequence context by molecular modeling. The development of a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions is described. A representative base-pairing and base-displaced conformation of [+ta]-B[a]P-N2-dG in the 5'-TGT sequence are used: (1) BPmi5, which has the B[a]P moiety in the minor groove pointing toward the base on the 5'-side of the adduct, and (2) Gma5, which has the B[a]P moiety stacked with the surrounding base pairs and the dG moiety displaced into the major groove. The MD output structures are reasonable when compared to known NMR structures. Changes in DNA sequence context dramatically affect the biological consequences (e.g., mutagenesis) of [+ta]-B[a]P-N2-dG. Consequently, we also developed a MD-based free-energy perturbation (FEP) protocol to study DNA sequence changes. FEP involves the gradual "fading-out" of atoms in a starting structure (A) and "fading-in" of atoms in a final structure (B), which allows a realistic assessment of the energetic and structural changes when two structures A and B are closely related. Two DNA sequence changes are described: (1) 5'-TGT --> 5'-TGG, which involves two steps [T:A --> T:C --> G:C], and (2) 5'-TGT --> 5'-TGC, which involves three steps [T:A --> T:2AP --> C:2AP --> C:G], where 2AP (2-aminopurine) is included, because T:2AP and C:2AP retain more-or-less normal pairing orientations between complementary bases. FEP is also used to evaluate the impact that a 5'-TGT to 5'-UGT sequence change might have on mutagenesis with [+ta]-B[a]P-N2-dG. In summary, we developed (1) a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions to study B[a]P-N2-dG adducts in DNA duplexes, and (2) a MD-based free-energy perturbation (FEP) protocol to study DNA sequence context changes around B[a]P-N2-dG adducts.
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PMID:Free-energy perturbation methods to study structure and energetics of DNA adducts: results for the major N2-dG adduct of benzo[a]pyrene in two conformations and different sequence contexts. 1602 3

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a deltadeltaG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.
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PMID:Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions. 1609 91

Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5'-TGT and 5'-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy.dG is mutagenic inducing primarily targeted Fapy.G-->T transversions. In the 5'-TGT sequence mutational frequency of Fapy.dG was approximately 30%, whereas in the 5'-TGA sequence it was approximately 8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy.dG, though it also exhibited a similar context effect: 4-fold G-->T transversions (24% versus 6%) occurred in the 5'-TGT sequence relative to 5'-TGA. To investigate a possible structural basis for the higher G-->T mutations induced by both lesions when their 3' neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase beta, which is known to incorporate both dCTP (no mutation) and dATP (G-->T substitution) opposite 8-oxo-G. In pol beta, the syn-8-oxo-G:dATP pair showed greater stacking with the 3'-T:A base pair in the 5'-TGT sequence compared with the 3'-A:T in the 5'-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5'-TGT and 5'-TGA sequences. Similarly, syn-Fapy.G:dATP pairing showed greater stacking in the 5'-TGT sequence compared with the 5'-TGA sequence, while stacking for anti-Fapy.G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3'-A:T base pair in the 5'-TGA sequence might cause lower G-->T mutational frequencies in the 5'-TGA sequence compared to 5'-TGT. The corresponding lesions derived from 2'-deoxyadenosine, Fapy.dA and 8-oxo-dA, were not detectably mutagenic in the 5'-TAT sequence, and were only weakly mutagenic (<1%) in the 5'-TAA sequence context, where both lesions induced targeted A-->C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy.G-->T substitutions occur at a higher frequency than 8-oxo-G-->T and that Fapy.dA is very weakly mutagenic, as is 8-oxo-dA.
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PMID:Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. 1667 49