Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 °C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.
Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase η is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol η depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.DNA damage | DNA polymerase | translesion DNA synthesis | aromatic amines A romatic amines are known to be strong carcinogens. After metabolic activation, aromatic amines react as electrophilic arylnitrenium ions with nucleophilic functionalities of the DNA duplex. Preferred reaction sides are the amino groups of adenine and guanine and particularly the C8-position of guanine (1, 2). The latter reaction gives rise to the so called C8-bulky adduct lesions, which interfere with the replication process leading to mutations (2). Two types of aromatic amine lesions are known. The nonacetylated lesions depicted in Fig. 1A reduce the replication efficiency but are in general faithfully bypassed by highfidelity polymerases (3). In contrast, the acetylated derivatives ( Fig. 1B) block replicative polymerases (3, 4), but can be bypassed with special low-fidelity polymerases (5-9). Moreover, if these acetylated lesions are present in certain repetitive sequences, they are known to induce frameshift mutations (4,(10)(11)(12). Recently, in vivo experiments revealed that the low-fidelity Y-family polymerase η is able to replicate DNA containing acetylated aromatic amine lesions such as the acetylated analogue of 2-aminofluorene-dG (AF-dG), 2-acetylaminofluorene-dG (AAF-dG) (13). Replication through the lesion is even for Pol η very difficult and hence slow, but if it occurs it is typically error free.The distinct mutagenic properties of the acetylated and nonacetylated aromatic amine lesions are caused by their different conformational preferences. While the nonacetylated lesions exist both in syn and anti conformation (14, 15), the corresponding acetylated lesions seem to adopt the syn-conformation with high preference (10,16,17). Crystal structures of Ellenberger, Beese, and Patel, showing the nonacetylated AF-dG-lesion inside different polymerases, prove that the lesion is indeed bound in anti conformation, allowing Watson-Crick base pairing with an incoming dCTP (18)(19)(20). While bypass of the nonacetylated lesions by high-fidelity polymerases is rather well understood, the mechanism that allows low-fidelity polymerases such as Pol η to replicate...
Side‐stepping thermodynamics. In contrast to the traditional benzoin reaction, which can yield a mixture of homo and mixed adducts, only one regioisomer was obtained from the new cyanide‐catalyzed silyl benzoin reaction between acylsilanes and aldehydes (see scheme).
Flipped out: The cisplatin–(1,3‐GTG) adduct, a strongly cytotoxic DNA lesion generated by the action of cisplatin therapeutics, is partially bypassed by Y‐family DNA polymerase η. Crystal structure data on this DNA–protein complex explain the partial‐bypass process. The central dT unit of the lesion is turned out and blocks the movement of the enzyme along the DNA duplex.
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