DNA: Structure, Organisation & Replication

The monomeric units — deoxyadenylate, deoxyguanylate, deoxycytidylate and thymidylate — are held in polymeric form by 3′,5′-phosphodiester bonds, with the bases attached to the 2-deoxyribose by an N-glycosidic bond.

The informational content of DNA (the genetic code) resides in the sequence in which these monomers are ordered.

The polymer possesses a polarity: one end has a 5′-hydroxyl or phosphate terminal, the other a 3′-phosphate or hydroxyl terminal. Convention dictates that a single-stranded DNA sequence is written in the 5′ to 3′ direction. Note also that the phosphodiester backbone is negatively charged.

The B form is usually found under physiologic conditions (low salt, high degree of hydration).

One turn of B-DNA about the long axis contains 10 bp.
The distance spanned by one turn is 3.4 nm (34 Å) — so the rise is 3.4 Å per base pair.
The width (helical diameter) is 2 nm (20 Å).

The helix is right-handed, because as one looks down the double helix the base residues form a spiral in a clockwise direction. It has a major groove and a minor groove, and in the test tube double-stranded DNA can exist in at least six forms (A–E and Z).

The two strands are held in register by both hydrogen bonds between the purine and pyrimidine bases of the respective linear molecules AND by van der Waals and hydrophobic interactions between the stacked adjacent base pairs.

Note that hydrogen bonding is only half the answer — base stacking contributes at least as much, and it is the half candidates usually forget.

Chargaff observed that in DNA molecules the concentration of deoxyadenosine (A) nucleotides equals that of thymidine (T) nucleotides (A = T), while the concentration of deoxyguanosine (G) equals that of deoxycytidine (C) (G = C).

Three hydrogen bonds hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the A-T pair is held together by two hydrogen bonds.

Thus the G-C bonds are more resistant to denaturation, or strand separation — termed “melting” — than A-T-rich regions of DNA.

The two strands, each of which possesses a polarity, are ANTIPARALLEL: one strand runs in the 5′ to 3′ direction and the other in the 3′ to 5′ direction.

Within a particular gene:
The TEMPLATE strand — the strand whose sequence carries the genetic information, and the strand of DNA that is copied during RNA synthesis. It is sometimes called the noncoding strand.
The CODING strand — the opposite strand, so called because it matches the sequence of the RNA transcript, but containing thymine in place of uracil.

Note the point students most often miss: the template strand for each gene will not necessarily be the same strand of the DNA double helix.

The double-stranded structure of DNA can be separated into two component strands in solution by increasing the temperature or decreasing the salt concentration. Not only do the two stacks of bases pull apart, but the bases themselves unstack while still connected in the polymer by the phosphodiester backbone.

Concomitant with denaturation is an increase in the optical absorbance of the purine and pyrimidine bases — a phenomenon referred to as HYPERCHROMICITY OF DENATURATION.

Because of the stacking of the bases and the hydrogen bonding between the stacks, the double-stranded DNA molecule exhibits properties of a rigid rod and in solution is a viscous material that loses its viscosity upon denaturation.

The strands of a given molecule of DNA separate over a temperature range; the midpoint is called the melting temperature, or T_m.

Three things influence it:
1 · 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.
2 · Salt concentration. A 10-fold increase of monovalent cation concentration increases the T_m by 16.6 °C, by neutralizing the intrinsic interchain repulsion between the highly negatively charged phosphates of the phosphodiester backbone.
3 · Solvent. Formamide destabilizes hydrogen bonding between bases, thereby lowering the T_m — used in recombinant DNA work to separate strands at much lower temperatures, minimising the phosphodiester bond breakage that can occur on extended incubation at higher temperatures.

Separated strands of DNA will renature or reassociate when appropriate physiologic temperature and salt conditions are achieved; this reannealing process is often referred to as HYBRIDIZATION.

The rate of reassociation depends upon the concentration of the complementary strands. Renaturation requires base pair matching — which is precisely what makes it useful, because a labelled probe will bind only to a sequence complementary to it.

Southern blotting — DNA/DNA. Northern blotting — RNA/DNA. (Western blotting, by the same joke, detects protein with antibody.)

DNA exists in relaxed and supercoiled forms. Supercoiling arises in a covalently closed circle — or in a linear molecule anchored at both ends, as chromosomal DNA is — because the two strands cannot rotate freely about each other.

Negative supercoils are underwound and favour strand separation; positive supercoils are overwound and oppose it.

The nicking-resealing enzymes that relieve torsional strain.

The nicks are quickly resealed without requiring energy input, because of the formation of a high-energy covalent bond between the nicked phosphodiester backbone and the nicking-sealing enzyme. Contrast this with DNA ligase, whose resealing is ATP-dependent.

Among the classes of proteins involved in replication, topoisomerases relieve torsional strain that results from helicase-induced unwinding.

Dense spherical particles, approximately 10 nm in diameter, connected by DNA filaments, composed of DNA wound around an octameric complex of histone molecules.

Structure: the histone octamer is 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 surface of the histone octamer, protecting 145 to 150 bp of DNA and forming the nucleosome core particle.

Assembly: 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. H1 histones are the ones least tightly bound to chromatin and are therefore easily removed with a salt solution; neither histone H1 nor the nonhistone proteins are necessary for the reconstitution of the nucleosome core. H1 binds the linker DNA between core particles.

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.

Histones are a small family of closely related basic proteins — that is, highly positively charged, which is what allows them to bind the negatively charged phosphodiester backbone.

The human haploid genome consists of about 3 × 10⁹ bp and about 1.7 × 10⁷ nucleosomes. The length of each DNA molecule must be compressed about 8000-fold to generate the structure of a condensed metaphase chromosome.

At metaphase, mammalian chromosomes possess a twofold symmetry, with the identical duplicated sister chromatids connected at a centromere, the relative position of which is characteristic for a given chromosome.

Each sister chromatid contains one dsDNA molecule. During interphase the packing of the DNA molecule is less dense than in the condensed chromosome at metaphase; metaphase chromosomes are nearly completely transcriptionally inactive.

In metaphase chromosomes, the 30-nm chromatin fibers are also folded into a series of looped domains. Humans have 23 pairs of chromosomes: 22 autosomes and two sex chromosomes.

An adenine-thymine (A-T)-rich region containing repeated DNA sequences that range in size from 10² (brewers' yeast) to 10⁶ (mammals) base pairs.

Metazoan centromeres are bound by nucleosomes containing the histone H3 variant protein CENP-A and other specific centromere-binding proteins. This complex, called the KINETOCHORE, provides the anchor for the mitotic spindle. It thus is an essential structure for chromosomal segregation during mitosis.

The structures at the ends of each chromosome.

Telomeres consist of short TG-rich repeats. Human telomeres have a variable number of repeats of the sequence 5′-TTAGGG-3′, which can extend for several kilobases.

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 the length of the telomere.

Since telomere shortening has been associated with both malignant transformation and aging, this enzyme has become an attractive target for cancer chemotherapy and drug development.

During a round of replication, each of the two strands of DNA is used as a template for synthesis of a new, complementary strand.

Each daughter duplex therefore consists of one strand derived from the original parental DNA duplex and one strand that was newly synthesized.

This is what makes faithful transmission possible: since the genetic information resides in the order of the monomeric units within the polymers, there must exist a mechanism of reproducing or replicating this specific information with a high degree of fidelity.

At the origin of replication (ori) there is an association of sequence-specific dsDNA-binding proteins with a series of direct repeat DNA sequences.

In E. coli the oriC is bound by the protein dnaA; in eukaryotes the group of proteins is the origin recognition complex (ORC), and ORC homologs have been found in all eukaryotes examined. The ORE lies adjacent to an approximately 80-bp A+T-rich sequence that is easy to unwind, called the DNA unwinding element (DUE).

Replication then proceeds bidirectionally, generating “replication bubbles” with a fork at each end.

DNA polymerases only synthesize DNA in the 5′ to 3′ direction, and only one of the several different types of polymerases is involved at the replication fork. Because the DNA strands are antiparallel, the polymerase functions ASYMMETRICALLY.

On the leading (forward) strand, the DNA is synthesized continuously.
On the lagging (retrograde) strand, the DNA is synthesized in short (1–5 kb) fragments, the so-called OKAZAKI fragments — several Okazaki fragments (up to a thousand) must be sequentially synthesized for each replication fork.

The helicase acts on the lagging strand to unwind dsDNA in a 5′ to 3′ direction, and associates with the primase to afford the latter proper access to the template. This allows the RNA primer to be made and, in turn, the polymerase to begin.

The segments of DNA attached to an RNA initiator component are the Okazaki fragments. In mammals, after many Okazaki fragments are generated, the replication complex begins to remove the RNA primers, to fill in the gaps left by their removal with the proper base-paired deoxynucleotide, and then to seal the fragments of newly synthesized DNA by enzymes referred to as DNA ligases.

The chemistry of chain growth: the template dictates which deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in place while the 3′-hydroxyl group of the growing strand attacks and incorporates the new nucleotide into the polymer.