Unit 23 Question Bank
Structure: the octamer is two each of H2A, H2B, H3 and H4. The DNA is supercoiled in a LEFT-HANDED helix over the surface of the disk-shaped histone octamer; 1.75 superhelical turns are wrapped around it, protecting 145 to 150 bp and forming the nucleosome core particle.
Assembly: the (H3-H4)₂ tetramer can itself confer nucleosome-like properties on DNA; addition of two H2A-H2B dimers stabilizes the particle. H1 is NOT part of the core — it is the histone least tightly bound to chromatin, binds the linker DNA, and is not necessary for reconstitution of the nucleosome core.
Function: to condense DNA — the human haploid genome contains about 3 × 10⁹ bp and about 1.7 × 10⁷ nucleosomes, and must be compressed ~8000-fold into a metaphase chromosome. But histones also integrally participate in gene regulation: acetylation of lysines in the histone tails reduces their positive charge, decreases affinity for DNA and disrupts nucleosomal structure, giving transcription factors access.Harper's ch.35, pp.371–375
Why they are needed — the end-replication problem: DNA polymerases synthesise only 5′→3′ and cannot initiate a chain, so on the lagging strand the final RNA primer at the chromosome end is removed and the gap cannot be filled. Every round of replication would therefore shorten the chromosome. Telomeres are expendable non-coding repeats that absorb this loss.
Telomerase — a multisubunit RNA template-containing complex related to viral RNA-dependent DNA polymerases (reverse transcriptases) — is the enzyme responsible for telomere synthesis and thus for maintaining telomere length.
Clinical significance: telomere shortening has been associated with both malignant transformation and aging, so telomerase has become an attractive target for cancer chemotherapy and drug development. Most somatic cells lack it; most cancers reactivate it.Harper's ch.35, pp.374–375
Denaturation is accompanied by hyperchromicity of denaturation — an increase in the optical absorbance of the purine and pyrimidine bases, because the bases unstack — and by a loss of viscosity, since double-stranded DNA exhibits properties of a rigid rod.
Three influences:
• Base composition — DNA rich in G-C pairs, which have three hydrogen bonds, melts at a higher temperature than that rich in A-T pairs, which have two.
• Salt — a 10-fold increase of monovalent cation concentration increases the Tm by 16.6 °C, by neutralizing the intrinsic interchain repulsion between the negatively charged phosphates.
• Solvent — formamide destabilizes hydrogen bonding between bases, thereby lowering the Tm.
Separated strands renature when appropriate temperature and salt conditions are achieved; this reannealing is often referred to as HYBRIDIZATION, and it is the basis of Southern (DNA/DNA) and Northern (RNA/DNA) blotting.Harper's ch.34, p.361
It is a direct consequence of the base-pairing rule: since A pairs only with T and G only with C, each strand already contains the information needed to rebuild the other.
Mechanically: replication starts at an origin (ori) — bound by dnaA in E. coli, by the origin recognition complex (ORC) in eukaryotes — beside an ~80-bp A+T-rich DNA unwinding element (DUE), and proceeds bidirectionally as a replication bubble with a fork at each end.
Because DNA polymerases synthesise only 5′→3′ and the strands are antiparallel, the polymerase functions asymmetrically: the leading strand is synthesized continuously and the lagging strand in short 1–5 kb Okazaki fragments, each primed by RNA and later sealed by DNA ligase.Harper's ch.34, p.363 · Harper's ch.35, pp.381–385
The polymer
The monomeric units — deoxyadenylate, deoxyguanylate, deoxycytidylate and thymidylate — are held in polymeric form by 3′,5′-phosphodiester bonds, with each base attached to its 2-deoxyribose by an N-glycosidic bond. The informational content of DNA resides in the sequence in which these monomers are ordered.
Each strand possesses a polarity — a 5′ and a 3′ end — and by convention a sequence is written 5′→3′. The phosphodiester backbone is negatively charged.
The double helix
Two strands wind around a central axis as a right-handed helix — right-handed because as one looks down the double helix the base residues form a spiral in a clockwise direction. The B form is usually found under physiologic conditions (low salt, high degree of hydration); at least six forms (A–E and Z) exist in the test tube.
Dimensions of B-DNA: 10 bp per turn; 3.4 nm (34 Å) per turn, hence 3.4 Å rise per base pair; helical diameter 2 nm (20 Å). It has a major and a minor groove.
The two strands are ANTIPARALLEL — one runs 5′→3′ and the other 3′→5′.
Base pairing
A pairs with T through two hydrogen bonds, G with C through three. This explains Chargaff's observation that A = T and G = C, and it is required by the geometry: only a purine paired with a pyrimidine keeps the diameter constant. Harper's adds that restrictions on rotation about the phosphodiester bond, the favored anti-configuration of the glycosidic bond, and the predominant tautomers of the four bases allow A to pair only with T, and G only with C.
The stabilising forces — both of them
The two strands are held in register by both hydrogen bonds between the bases AND by van der Waals and hydrophobic interactions between the stacked adjacent base pairs. Base stacking is as important as hydrogen bonding, and it is the loss of stacking that produces hyperchromicity on denaturation.
Consequences of the structure
- G-C-rich DNA has a higher Tm, because of the third hydrogen bond — which is why replication origins sit beside A+T-rich unwinding elements.
- The grooves allow sequence recognition without unwinding — the edges of the base pairs are exposed at the floor of the major groove, which is where the helix-turn-helix, zinc finger and leucine zipper motifs bind.
- The base-pairing rule is the copying mechanism: each strand contains the information to rebuild the other, which is what makes semiconservative replication possible.
The problem
Human genomic DNA, if extended end-to-end, would be metres in length, yet must fit within a nucleus only microns in diameter — Harper's puts the total at thousands of times the diameter of the cell nucleus. The solution is a hierarchy of folding, each level built on the last.
Level 1 — the nucleosome
Electron microscopic studies have demonstrated dense spherical particles called nucleosomes, approximately 10 nm in diameter and connected by DNA filaments. Each consists of DNA wound around an octameric complex of histone molecules — two each of H2A, H2B, H3 and H4.
In the nucleosome, the DNA is supercoiled in a LEFT-HANDED helix over the surface of the disk-shaped histone octamer. 1.75 superhelical turns of DNA are wrapped around the octamer, protecting 145 to 150 bp and forming the nucleosome core particle.
Assembly is ordered: the (H3-H4)₂ tetramer itself can confer nucleosome-like properties on DNA and thus has a central role in the formation of the nucleosome; the addition of two H2A-H2B dimers stabilizes the primary particle.
H1 is not part of the core. It is the histone least tightly bound to chromatin, easily removed with a salt solution, and neither H1 nor the nonhistone proteins are necessary for reconstitution of the nucleosome core; it binds the linker DNA between core particles.
Level 2 — chromatin
Strings of nucleosomes form along the linear sequence of genomic DNA to form chromatin, which itself can be more tightly packaged and condensed, ultimately leading to the formation of the chromosomes. The 30-nm fibre is folded into looped domains anchored to the nuclear matrix.
Level 3 — the chromosome
At metaphase, mammalian chromosomes possess a twofold symmetry, with the identical duplicated sister chromatids connected at a centromere. Each sister chromatid contains one dsDNA molecule. The overall compression is about 8000-fold; the human haploid genome of 3 × 10⁹ bp is held in about 1.7 × 10⁷ nucleosomes.
Two specialised regions complete the structure. The centromere is an A-T-rich region of repeated DNA bound by nucleosomes containing the histone H3 variant CENP-A; this complex, the kinetochore, provides the anchor for the mitotic spindle. The telomeres are short TG-rich repeats, in humans 5′-TTAGGG-3′, maintained by telomerase.
Why packaging is regulation
Harper's is explicit that condensation is not the whole story: the histones also integrally participate in gene regulation; indeed histones contribute importantly to all DNA-directed molecular transactions.
The mechanism is electrostatic. Histones are basic — positively charged — and DNA is negatively charged. Acetylation on lysine residues in the amino-terminal tails reduces the positive charge of these tails, decreases the binding affinity of histone for DNA, and disrupts nucleosomal structure, allowing access of transcription factors to cognate regulatory DNA elements and enhancing binding of the basal transcription machinery to the promoter. Histone deacetylation has the opposite effect.
A second layer is methylation of deoxycytidine residues in the sequence 5′-mCpG-3′, which silences genes — in mouse liver, only the unmethylated ribosomal genes can be expressed, and many animal viruses are not transcribed when their DNA is methylated.
A gene wrapped tightly in nucleosomes is off; one whose nucleosomes have been loosened or evicted is available. Packing and permission are the same thing. Metaphase chromosomes are nearly completely transcriptionally inactive — the extreme case of the same principle.