Abstract:Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We als… Show more
“…Shown in Figure 7A are emission spectra for a single-stranded 16-mer containing a single 2AP at its 5′-end (oligo 3, Table 1), as a function of [AGT]. The emission maxima at 369 nm are similar to values reported for other 2AP-labeled DNAs (47,48), while the intensity increase with AGT binding is like that seen with other proteins that stabilize extrahelical base conformations in DNA (49,50). Figure 7.Base-flipping detected by 2AP fluorescence.…”
O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee–von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (∼0, ∼30 and ∼60°) that are consistent with models in which each protein induces a bend of ∼30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.
“…Shown in Figure 7A are emission spectra for a single-stranded 16-mer containing a single 2AP at its 5′-end (oligo 3, Table 1), as a function of [AGT]. The emission maxima at 369 nm are similar to values reported for other 2AP-labeled DNAs (47,48), while the intensity increase with AGT binding is like that seen with other proteins that stabilize extrahelical base conformations in DNA (49,50). Figure 7.Base-flipping detected by 2AP fluorescence.…”
O6-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee–von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (∼0, ∼30 and ∼60°) that are consistent with models in which each protein induces a bend of ∼30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.
“…A satisfactory fit was obtained with 4 lifetime components, ranging from ∼100 ps to nearly 10 ns, demonstrating the large conformational heterogeneity explored by the 2-Ap residues at each position (Table 1). The longest-lived component τ 4 that likely corresponded to conformations where 2-Ap was either unstacked or extrahelical (63,64) varied from 6.1 to 9.6 ns. The shortest τ 4 values likely resulted from conformational dynamics of the 2-Ap residues in their excited state, which may drive them from an unstacked to a stacked conformation.…”
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.
“…If a cleft capable of binding an extrahelical DNA base is sufficient for binding FAM or other fluorophores, then a large family of mechanistically-related enzymes may be prone to interactions with these residues. Related enzymes include the human and bacterial O 6 -alkylguanine alkyltransferases (AGTs), the yeast and bacterial alkyltransferase-like proteins (ATLs), human alkyladenine glycosylase (hAAG), 8-oxoguanine DNA glycosylase (hOGG), human and bacterial uracil-DNA glycosylases (UDG or UNG), oxidative DNA/RNA dealkylases such as E. coli AlkB and its human homologue ABH2, and a large number of bacterial host-restriction DNA methyltransferases such as EcoRI methylase [12,13,51-53]. …”
Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ~40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC50 ~ 76μM) and repair of DNA containing an O6-methylguanine residue (IC50 ~ 63μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.
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