DNA-binding proteins resemble their mismatches.

DNA-bound proteins are ubiquitous in biology. The ability to bind these proteins to specific DNA sequences with high affinity is often central to their function, and it is not uncommon for a single mutation to affect the ability of a protein to bind DNA. It is surprising, then, to find that many DNA-binding proteins can bind more tightly to sequences that have been engineered to involve a type of single-nucleotide transformation called a mismatch. But that’s really it and others.1report in Nature.

There is a significant difference between a mutation and a mismatch, even though both involve changing the identity of a nucleotide. A mutation occurs on both strands of the DNA double helix. This means that the base-pairing between the DNA bases on each strand is maintained. A mismatch, however, occurs on only one strand, and therefore the normal base-pairing is eliminated. In normal base-pairing, there is an adenine (A) base at one end of the DNA duplex pair with thymine (T) on the complementary strand, and guany (G) bases with cytosine (C) – so from A-T The change is a pairwise mutation in CG pairs, while the change in AC is a mismatch. Because mismatches are not pair-wise, they can distort the overall structure of DNA more easily than mutations (Fig. 1).

Figure 1 | Re-shaping DNA. The DNA double helix consists of pairs of DNA bases: adenine (A) pairing with one strand pair with thymine (T) and guanine (G) bases with cytosine (C) on the other. a, A double helix may exist in a series of shapes, called a conformal ensemble. In this simple schematic, the dominant construct adopting wild-type DNA is at the forefront, and the slight conformity that it can transiently adopt is the beige shadow behind. B, A mutation transforms one pair into another pair (eg AT to C – G). This mutation is unlikely to alter the ensemble of possible conformations (although it occasionally occurs; not shown). C, In a mismatch, just one base is replaced, interrupting the base-pairing (for example AT AC can be formed). This disruption is likely to lead to a change in the conformal assembly. D, Protein binding (red) can also alter DNA homology. Afek and others.1 Report that 10% of mismatches flank DNA into configurations that are more similar to protein-bound DNA than wild-type versions, making it easier for proteins to bind.

It would be reasonable to assume that DNA deformations would impair protein binding, but in fact this shape may contribute to binding specificity through a mechanism known as readout. In simple terms, shape readout is the ability of proteins to indirectly identify specific DNA sequences by their specific 3D shapes.2,3. This is in contrast to their ability to directly identify specific sequences by specific chemical groups present in each base pair, a mechanism known as base readout. DNA is often considered to be the same size regardless of its sequence, but the size readout works because it is not strictly true. Each sequence has a preferred set of conformations (called analogues) and can be more or less easily bent in different ways. Taking advantage of this, a protein that needs to bind to a specific sequence can try to bend any sequence it encounters in a way that would be most compatible with its intended target. Because bending DNA has an energetic cost, this mechanism leads to a decrease in binding affinity.

Shape readout plays a role in many protein-DNA interactions.2,3, But its true energetic cost has been difficult to study, because to do so one would have to maintain the shape of the DNA molecule without creating its sequence. Afek and others. Realized that mismatches, being small changes in a sequence that lead to large changes in size, provide a unique way to study this phenomenon.

Working with mismatched DNA is not trivial, especially in high-throughput conditions, as many standard molecular-biology techniques predict that DNA is completely base-coupled. The authors therefore developed what they call a saturation mismatch-binding assay (SaMBA), which determines the amount of binding of a protein to every possible single-nucleotide mismatch in a particular DNA sequence. In short, they constructed a microchip with single strands of DNA encoding every possible single-nucleotide variant of the consensus sequence. Each strand was placed on a known coordinate on the chip. They then allowed a second, complementary DNA strand to flow over the array. The complementary DNA hybridizes with each strand printed on the chip, creating duplex DNA with every possible mismatch. Finally, the authors added fluorescently labeled proteins and overviewed its bonding with DNA using microscopy. They ran the assay using 22 different sets of DNA arrays and proteins.

SaMBA not only stated that it is possible for mismatches to improve DNA-protein binding, but also that it is relatively common for them to do so. The nearly 10% mismatch that Afek and co-workers analyzed increased the affinity with which a protein is associated with a sequence that contains at least one such sequence for each protein. For some proteins, the most effective mismatch occurred in the natural target sequence, causing the protein to bind even more tightly with that sequence. For others, the most effective mismatch occurred in a non-target sequence, and bound the protein to that order at a level comparable to the natural target. In both cases, the same mechanism is predominant: mismatch pays the energetic cost of deforming DNA so that the protein does not.

Note that to actually improve the bond, the mismatch would have to deform the DNA in the same way as the protein via the shape-readout mechanism. Deforming the DNA in a different way would weaken the binding. Mismatches should not interfere with any chemical contacts between proteins and DNA – although the authors found that mismatched contacts can sometimes present favorable contacts.

The work of Afek and colleagues broadens our understanding of how proteins bind to DNA, and highlights the importance of DNA conformance in the process. In the future, perhaps nucleotides that do not occur in nature4 It can be used in SaMBA to fully examine the array of DNA conjugates, in the same way that unnatural amino acids have been used to investigate subtle changes in protein biophysics.5. SaMBA can potentially be adapted to find DNA-binding proteins that bind mismatched or chemically modified targets; Such proteins would be difficult to find in other ways. Given that approximately one-third of transcription factors (a major class of DNA-binding proteins that regulate gene expression) have no known target sequence in humans6, This can be a productive line of investigation.

Broadly, it suggests that improving mismatched marriage bonds can often have implications for diseases such as cancer. Even a transient mismatch in the genome can cause a transcription factor to bind in the wrong place, where it can potentially misregulate a gene and place the cell in a cancer transcriptional state that mismatch. Persists even after repair. Given its temporary root cause, it would be difficult to study or confirm this idea. But the apparent tendency for mismatches to improve bonding makes such a mechanism worth pondering.

Leave a Reply