DNA Structure, Replication, and Repair

Core Concepts

DNA structure

DNA (deoxyribonucleic acid) is a polymer of nucleotides. Each nucleotide has three components:

• A five-carbon sugar: deoxyribose

• A phosphate group

• A nitrogenous base: adenine (A), thymine (T), cytosine (C), or guanine (G)

Nucleotides are linked by phosphodiester bonds between the 3' carbon of one sugar and the 5' carbon of the next, forming a sugar-phosphate backbone. The bases project inward.

DNA is double-stranded. The two strands are:

• Antiparallel: One strand runs 5'→3'; the other runs 3'→5'. The 5' end has a free phosphate group; the 3' end has a free hydroxyl group.

• Complementary: A always pairs with T (via 2 hydrogen bonds); G always pairs with C (via 3 hydrogen bonds).

• Helical: The two strands wind around each other, forming a double helix. The sugar-phosphate backbones are on the outside; the bases are stacked on the inside, like rungs of a spiral ladder.

The base-pairing rules are the key to DNA's function: they allow one strand to serve as a template for building the complementary strand, enabling accurate replication.

DNA as genetic material: The Hershey-Chase experiment (1952) used radioactive isotopes to demonstrate that DNA — not protein — enters bacterial cells during phage infection and directs viral reproduction.

Semiconservative replication

DNA replication is semiconservative: each of the two parental strands serves as a template for the synthesis of a new complementary strand. After replication, each daughter DNA molecule consists of one original (parental) strand and one newly synthesized strand. This was demonstrated by the Meselson-Stahl experiment, which used density-gradient centrifugation of DNA labeled with heavy nitrogen (15N).

The replication process

Origins of replication: Replication begins at specific DNA sequences called origins of replication. Prokaryotes typically have a single origin; eukaryotes have multiple origins per chromosome. The DNA unwinds at each origin, forming a replication bubble with two replication forks (one at each end of the bubble).

Key enzymes: Helicase unwinds DNA. SSBs prevent re-annealing. Topoisomerase relieves supercoiling. Primase synthesizes RNA primer (DNA polymerase needs a free 3'-OH). DNA polymerase adds nucleotides 5'→3'. Ligase seals fragments.

Leading and lagging strands

Because DNA polymerase can only synthesize in the 5'→3' direction, and the two template strands are antiparallel, the two new strands are synthesized differently:

• Leading strand: The template strand runs 3'→5', so the new strand can be synthesized continuously in the 5'→3' direction toward the replication fork. Only one primer is needed.

• Lagging strand: The template strand runs 5'→3', so the new strand must be synthesized in the opposite direction — away from the replication fork — in short fragments. Each fragment requires a new RNA primer. These fragments are called Okazaki fragments. DNA polymerase I removes the RNA primers and replaces them with DNA; ligase seals the gaps between fragments.

DNA organization

• Prokaryotes: Typically a single, circular chromosome located in the nucleoid region. DNA is supercoiled and associated with some proteins, but not organized into the highly structured chromatin of eukaryotes. Many prokaryotes also have smaller circular DNA molecules called plasmids.

• Eukaryotes: Multiple linear chromosomes located in the nucleus. DNA is wrapped around histone proteins to form nucleosomes — bead-like structures that are the fundamental unit of chromatin packaging. Chromatin is further folded and condensed, with the degree of condensation varying throughout the cell cycle (highly condensed during mitosis, less condensed during interphase).

Proofreading and repair

DNA replication is remarkably accurate — typically only about one error per billion nucleotides copied. This accuracy results from:

1. Proofreading: DNA polymerase's 3'→5' exonuclease activity removes mismatched nucleotides and replaces them.

2. Mismatch repair: After replication, enzymes detect and repair mismatched base pairs that escaped proofreading.

3. Nucleotide excision repair: Enzymes remove and replace damaged DNA segments (e.g., thymine dimers caused by UV radiation).

4. Other repair pathways: Base excision repair, double-strand break repair, and others address different types of damage.

When repair mechanisms fail, mutations accumulate. Mutations in DNA repair genes themselves dramatically increase cancer risk — for example, mutations in mismatch repair genes are associated with certain hereditary colorectal cancers.

ELI Example

DNA replication is like photocopying a book by tearing it down the middle. Each half-page serves as a template — wherever you see the top half of an "A," you print a "T" on the new matching half; wherever you see "G," you print "C." After photocopying, you have two complete books, each with one old half and one new half. The photocopier is incredibly accurate, with a built-in spell-checker that catches errors as it goes. If a page is damaged (mutated), repair crews can cut out the damaged section and patch in a correct copy based on the undamaged strand.

Do Not Confuse

High-Yield Memory Anchors

• A-T (2 H-bonds), G-C (3 H-bonds). Antiparallel strands.

• Replication = semiconservative (one old, one new strand per molecule).

• Leading = continuous. Lagging = Okazaki fragments, joined by ligase.

• DNA polymerase: 5'→3' synthesis; 3'→5' proofreading.

• Primase makes RNA primer. Helicase unwinds. Ligase seals.

Quick Check

Q1 (Foundational): List the four nitrogenous bases in DNA. Which pairs with which, and how many hydrogen bonds form between each pair?

Q2 (Application): A segment of one DNA strand has the sequence 5'-ATCGGCTA-3'. What is the sequence of the complementary strand (indicating 5' and 3' ends)?

Q3 (Comparison/Reasoning): Compare the synthesis of the leading and lagging strands during DNA replication. Why must the lagging strand be synthesized as Okazaki fragments?

Quick Check Answers

A1: Adenine (A) pairs with thymine (T) — 2 hydrogen bonds. Guanine (G) pairs with cytosine (C) — 3 hydrogen bonds.

A2: The complementary strand runs antiparallel: 3'-TAGCCGAT-5'. Written in the standard 5'→3' direction: 5'-TAGCCGAT-3'. (A pairs with T, T with A, C with G, G with C, and so on.)

A3: Both strands are synthesized in the 5'→3' direction by DNA polymerase. The leading strand is synthesized continuously because its template runs 3'→5', so the polymerase can simply add nucleotides as the fork opens. The lagging strand's template runs 5'→3', which would require synthesis in the 3'→5' direction — which DNA polymerase cannot do. Instead, the lagging strand is synthesized discontinuously as Okazaki fragments. Each fragment is primed with an RNA primer and synthesized 5'→3' away from the fork. The fragments are later joined by DNA ligase after primers are replaced with DNA. The lagging strand requires multiple priming events, while the leading strand requires only one.

Chapter Summary

DNA: antiparallel double helix, A-T and G-C base pairs. Semiconservative replication: each new DNA = one old + one new strand. Leading strand = continuous; lagging = Okazaki fragments joined by ligase. DNA polymerase proofreads (3'→5' exonuclease). Prokaryotic DNA: circular, nucleoid. Eukaryotic: linear, chromatin.

Common Mistakes

Mistake: "DNA polymerase synthesizes both strands continuously."

Reality: Because DNA polymerase can only synthesize 5'→3', and the template strands are antiparallel, the lagging strand must be synthesized discontinuously as Okazaki fragments.

Mistake: "DNA polymerase can start a new strand from scratch."

Reality: DNA polymerase requires a free 3'-OH group. Primase must first synthesize an RNA primer to provide that 3'-OH end.

Mistake: "The two strands of DNA run in the same direction."

Reality: The strands are antiparallel — one runs 5'→3', the other runs 3'→5'. This antiparallel arrangement is essential for how replication works.

Mistake: "Mutations are always caused by replication errors."

Reality: Mutations also arise from environmental factors (radiation, chemical mutagens) and spontaneous chemical changes (e.g., deamination of cytosine to uracil). Many mutations are repaired before they become permanent.