DNA inside cells is wrapped around a complex of histone proteins to yield higher-order structures known as nucleosomes and chromatin1 (Fig. 1). Inspection of the nucleosome structure2 might lead to the suspicion that substantial regions of DNA that face the histone core will be protected from reaction with small molecules such as anticancer drugs and mutagens. Contrary to that expectation, on page 79 of this issue, Boger and coworkers present data showing that the natural products yatakemycin and duocarmycin SA alkylate nucleosomal DNA, even at 'difficult' regions where target sites on the DNA face the protein core, with efficiencies and sequence specificities nearly identical to those seen for their reactions with 'naked' (protein-free) DNA3. This work offers a striking new demonstration that the dynamic nature of histone-DNA interactions can allow small molecules (with a molecular mass of less than 1,000 Da) to access target sites on the double helix that seem to be completely masked in the static structure of the nucleosome.

Figure 1: DNA in cells is associated with histone proteins to yield higher-order structures known as the nucleosome core particle and chromatin.
figure 1

Ann Thomson

Natural products such as duocarmycin can access, recognize and alkylate protein-associated sites in the nucleosome core particle.

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Accurate 'readout' (transcription) and copying (replication) of DNA is absolutely required for the normal operation of cells. It is not unexpected, then, that chemical modification of cellular DNA has profound biological consequences and represents an important area of study in the fields of medicinal chemistry, toxicology and biotechnology4,5. In studies aimed at elucidating the chemical processes underlying the biological activity of DNA-damaging drugs and mutagens, it is common to use isolated DNA fragments to model the cellular substrate6,7,8.

Of course, DNA inside the nucleus of mammalian cells differs from naked DNA, in that it is associated with proteins to yield higher-order structures. The first level of this higher-order organization is the nucleosome core particle, in which a 147–base pair (bp) stretch of DNA is wrapped around a disc-shaped protein complex consisting of four pairs of histone proteins (eight total proteins). The 1.7 turns of left-handed superhelix imposed on the DNA when it is incorporated into a nucleosome causes considerable curvature of about 45° per helical turn and yields grooves on the double helix that are deep and narrow facing toward the protein core and wide and flattened facing away2. There is overwinding of the helix in the central three turns and underwinding in the remainder of the DNA. Proteins in the histone core make close contact with 14 segments of the DNA minor groove and insert an arginine residue into the groove at each of these sites. In the cell nucleus, nucleosome core particles are connected by 20- to 60-bp regions of linker DNA and, in partnership with the linker histone H1, condense to form chromatin1 (Fig. 1). In this way, 2 meters of genomic DNA is compacted into a nuclear space less than 1 picoliter (1 × 10−12 liters) in volume.

Inspection of the static X-ray structure of the nucleosome2 might lead to the expectation that substantial regions of nuclear DNA will be completely protected from reaction with electrophilic alkylating agents such as anticancer drugs or mutagens. Notably, this is usually not the case. In fact, reactions of alkylating agents with nucleosomal DNA typically mirror those seen with naked DNA, showing very similar sequence preferences and only modestly (2- to 3-fold) decreased adduct yields9,10,11.

Yatakemycin and duocarmycin SA are potential anticancer agents with extremely potent biological activity. These natural products associate noncovalently with AT tracts in duplex DNA and alkylate at the N3 position of adenine residues. Both noncovalent binding and alkylation are highly dependent on the size and shape of the DNA minor groove at the target sites, making it especially interesting to investigate whether these compounds can recognize and covalently modify the rather distorted DNA found in the nucleosome.

In this issue, Boger and coworkers3 report that alkylation of DNA in a nucleosome core particle by yatakemycin and duocarmycin SA closely parallels their reactions with naked DNA. In this case, the sequence preferences are essentially identical, and alkylation yields decrease by a factor of only 1.4 to 1.8 in nucleosomal DNA versus naked DNA. The compounds efficiently alkylate 'difficult' sites that seem to be completely obscured by association with histone proteins in the X-ray structure of the nucleosome core particle2. Efficient reaction at these sites is especially remarkable given that, prior to DNA alkylation, yatakemycin and duocarmycin SA must 'find' and then noncovalently associate with fairly large (approximately 5-bp) binding sites. Access to 'difficult', protein-bound sites in nucleosomal DNA may be especially favored for alkylating agents whose unproductive decomposition reactions (such as hydrolysis) are slow relative to the rates of histone-DNA dissociation and DNA alkylation10. In essence, such agents have the ability to 'wait around' for the transient dissociation of the DNA-histone complexes that may allow them to recognize and alkylate 'normal' B-form DNA. Alternatively, the reaction could proceed via an induced-fit mechanism in which the alkylating agent induces distortion of DNA in the histone-DNA complex into a B-form-like structure, with the wider, shallower minor groove required for DNA modification by this class of agents. Notably, additional experiments show that the alkylation of nucleosomal DNA by these potential anticancer agents does not significantly disrupt the structural integrity of the nucleosome core particle, despite the likelihood12 that the DNA at these alkylation target sites is considerably distorted relative to that in the native nucleosome core particle.

Overall, these studies highlight the plasticity of the DNA-protein interactions in the nucleosomal core particle and show that the natural products yatakemycin and duocarmycin SA can access, recognize and alkylate large (5-bp), protein-associated binding sites in the nucleosome core particle. The core particle evidently can then adjust its structure to accommodate the chemically modified DNA. From a practical perspective, the authors correctly note that the similarities between the reactions observed for nucleosomal and naked DNA seen in these studies affirm the longstanding idea that in vitro experiments using naked (protein-free) DNA represent a valid and indispensable approach for predicting the site and structure of DNA damage that will be caused by small organic molecules in vivo. Such studies are a central part of important efforts directed at understanding the relationships between the chemical structure of DNA damage and the resulting biological response.