lecture 18: supercoiling, dna structure, lk

Proteins recognize specific DNA sequences through a combination of hydrogen bond donors and acceptors exposed in the major and minor grooves of the DNA helix. The pattern of hydrogen bonding is not just unique for an adenine-thymine (AT) pair but also depends on the directionality (e.g., AT versus TA), meaning that flipping a base pair can create a different pattern of donors and acceptors. This allows proteins to achieve sequence-specific interactions. In the major groove, proteins can discriminate among AT, TA, GC, and CG base pairs, while in the minor groove, discrimination is possible mainly between AT/TA and GC/CG pairs. Remarkably, as few as four base pairs are sufficient to create a unique binding site in the entire genome. Expanding the recognition sequence makes the binding even more specific. The search for new hydrogen bonding contacts in DNA recognition is relatively constrained because only a short list of amino acids can act as reliable hydrogen bond donors or acceptors. Common amino acids found in DNA-binding domains include asparagine (Asn), glutamine (Gln), glutamic acid (Glu), lysine (Lys), and arginine (Arg). These amino acids share key features: they often have longer side chains terminating in polar functional groups, enabling them to form hydrogen bonds with nitrogenous bases. Side chains terminate in groups capable of forming hydrogen bonds (e.g., amides in Asn/Gln, carboxylates in Glu, guanidinium in Arg). These groups align with the hydrogen bond donors/acceptors in the DNA grooves. Longer side chains (e.g., Arg, Gln, Lys) reach into the grooves to access base-specific interactions. Positively charged residues (Arg, Lys) interact with DNA’s negatively charged phosphate backbone, stabilizing binding. This is particularly critical in the minor groove, where narrowed regions enhance negative charge density.

Major Groove: Exposes distinct hydrogen bond patterns for each base pair (AT, TA, GC, CG). Asn and Gln discriminate between AT and TA by recognizing the unique arrangement of donors/acceptors.

Minor Groove: Narrower and less chemically diverse, limiting discrimination to AT/TA vs. GC/CG.

Proteins interact with DNA through specific structural motifs that allow for effective binding. Some common motifs include leucine zippers, zinc fingers, and helix-turn-helix structures. All of these motifs involve DNA-binding sites. DNA-binding proteins can bind either sequence-specifically, meaning they recognize and bind specific DNA base sequences, or non-sequence-specifically, meaning they primarily interact with the phosphate backbone of DNA rather than the bases themselves.

To begin understanding DNA compaction in eukaryotic cells, it’s important to recognize that DNA is not left freely floating. Instead, it is efficiently packaged into nucleosomes, where a segment of DNA wraps around histone proteins. Histones are small, positively charged proteins, rich in lysine and arginine residues. The abundance of these basic amino acids is crucial because their positive charges interact favorably with the negatively charged phosphate backbone of DNA, promoting tight wrapping. Each nucleosome core particle is made up of eight histone proteins—specifically, two copies each of H2A, H2B, H3, and H4—and about 146 base pairs of DNA are wrapped around this histone core. These nucleosome structures are found in the chromatin of all eukaryotic cells, serving not only to compact DNA but also to facilitate further supercoiling and higher-order structural organization.

Chemical modifications, such as acetylation or methylation of histone tails, can modulate how tightly DNA is wound around histones, influencing whether a gene is actively transcribed or silenced. The N-terminal “tails” of histones protrude from the nucleosome and are crucial in regulating DNA accessibility. These tails contain many lysine residues, which are subject to post-translational modifications. For example, acetylation of lysine residues neutralizes their positive charge, reducing histone-DNA interaction strength and making the DNA less tightly packed. This change increases the accessibility of DNA for processes like transcription.

In addition to histone modifications, long non-coding RNAs (lncRNAs) can influence DNA compaction. These RNAs do not code for proteins but can act as scaffolds or guides to recruit chromatin-modifying complexes to specific genomic regions, thereby impacting gene regulation by changing the local DNA accessibility.

There are different types of DNA duplexes based on environmental conditions and the types of nucleic acids involved. The A form of DNA typically occurs in DNA-RNA or RNA-RNA duplexes and is right-handed with approximately 11 base pairs per helical turn. The B form is the most common structure for DNA in cells, also right-handed, but with about 10.5 base pairs per turn. Less commonly, DNA can adopt a Z form, which is left-handed; however, the precise number of base pairs per turn in Z-DNA is not required knowledge for this course.

Normal B-form DNA parameters serve as a reference point for understanding DNA structure. B-form DNA, the most common form of DNA found in cells, has about 10.5 base pairs per helical turn when relaxed. The height of one full helical turn — called the pitch — is approximately 35.7 angstroms (Å). B-form DNA exhibits distinct major and minor grooves, which are critical for protein-DNA interactions. Importantly, any deviation from the standard 10.5 base pairs per turn introduces torsional stress into the DNA molecule. Cells manage this torsional stress through the phenomenon of supercoiling.

Supercoiling arises when the linking number (Lk) of DNA — a measure of how many times the two strands wrap around each other — changes. In a relaxed circular DNA molecule, the linking number is stable; for example, Lk = 200 for a DNA circle of about 2100 base pairs. If the DNA’s linking number is altered, either by adding or removing helical turns, the molecule compensates by forming supercoils. If two turns are removed (ΔLk = -2), the linking number decreases to 198, leading to the formation of negative supercoils. These left-handed supercoils relieve helical tension and make the DNA easier to unwind, which is beneficial for processes like transcription and replication. Negative supercoiling is the dominant form found in cells, especially in both bacteria and eukaryotes. Conversely, if two turns are added (ΔLk = +2), the linking number increases to 202, resulting in positive supercoils. These right-handed coils make the DNA more tightly wound and harder to separate.

Two DNA molecules that share the same nucleotide sequence but have different linking numbers are referred to as topoisomers — they are topologically distinct forms of DNA.

Cells regulate DNA supercoiling using enzymes called topoisomerases. Type I topoisomerases alter the linking number in steps of one by cutting a single strand of the DNA duplex, allowing the intact strand to rotate freely to relieve superhelical tension. This type of enzyme does not require ATP and can relax both positive and negative supercoils. Think of Type I topoisomerases as gentle tools that act like pressure release valves for the DNA. Type II topoisomerases, on the other hand, change the linking number in steps of two. They cut both strands of the DNA duplex, pass another segment of duplex DNA through the break, and then reseal it. Type II enzymes require ATP to perform these energy-intensive operations. They are capable of relaxing both types of supercoils and, importantly, can introduce negative supercoils — a process that is particularly vital in prokaryotic cells, where specialized Type II topoisomerases like DNA gyrase are present. In short, Type I topoisomerases perform subtle, ATP-independent modifications, while Type II topoisomerases act more forcefully and require energy input.

In summary, if the change in linking number (ΔLk) is less than zero, the DNA becomes negatively supercoiled (underwound). If ΔLk is greater than zero, the DNA becomes positively supercoiled (overwound). Type I topoisomerases cut one strand and change the linking number by ±1 without using ATP, whereas Type II topoisomerases cut both strands, change the linking number by ±2, require ATP, and can introduce negative supercoils into the DNA.