DNA Structure and Replication

DNA Structure — The Molecular Basis of Heredity

Part 1 of 7

Understanding DNA replication requires a thorough understanding of DNA structure. The double helix model, proposed by Watson and Crick in 1953 (based on X-ray crystallography data from Rosalind Franklin and Maurice Wilkins), remains one of the most important discoveries in biology.

Nucleotide Structure

DNA is a polymer of nucleotides. Each nucleotide has three components:

1. Deoxyribose sugar (5-carbon sugar lacking an -OH group at the 2' carbon)
2. Phosphate group (attached to the 5' carbon of the sugar)
3. Nitrogenous base (attached to the 1' carbon of the sugar)

The four DNA bases:

Chargaff's Rules: In any DNA molecule, the amount of A equals the amount of T, and the amount of G equals the amount of C. This 1:1 ratio (A=T, G=C) was critical evidence for the base-pairing model.

Key structural features of the double helix:

• Two antiparallel strands (one runs 5' → 3', the other 3' → 5')
• Sugar-phosphate backbones on the outside; bases face inward
• Bases held together by hydrogen bonds (A-T: 2 H-bonds; G-C: 3 H-bonds)
• The helix has a major groove and a minor groove (important for protein-DNA interactions)
• One complete turn = 10 base pairs = 3.4 nm
• Diameter = 2 nm

Checkpoint — DNA Structure

DNA Packaging in Eukaryotes

Human cells contain about 6.4 billion base pairs of DNA (~2 meters per cell if stretched out). This must be packaged into a nucleus only ~6 $\mu$m in diameter — a compaction ratio of ~10,000:1.

Packaging hierarchy:

1. Nucleosome — 147 bp of DNA wraps ~1.65 times around a histone octamer (2 copies each of H2A, H2B, H3, H4); the fundamental unit of chromatin (11 nm "beads on a string")
2. Linker histone H1 binds between nucleosomes, helping them pack into a 30 nm fiber
3. Looped domains — 30 nm fiber forms loops of 30,000-100,000 bp attached to a protein scaffold
4. Heterochromatin — maximally condensed, transcriptionally inactive
5. Metaphase chromosome — highest compaction (~10,000-fold), visible under a light microscope

Epigenetics and Histones: Chemical modifications to histones (acetylation, methylation, phosphorylation) alter chromatin structure and gene accessibility. Histone acetylation (by HATs) generally OPENS chromatin (euchromatin, active). Histone deacetylation (by HDACs) CLOSES it (heterochromatin, silenced).

Key Terms — DNA Structure

Exit Ticket — DNA Structure

Semiconservative Replication

Part 2 of 7

Three models for DNA replication were proposed:

1. Conservative — the original double helix remains intact; a completely new copy is made
2. Semiconservative — each new double helix consists of one original (parental) strand and one new strand
3. Dispersive — parental and new DNA are interspersed throughout both strands

The Meselson-Stahl experiment (1958) elegantly determined which model is correct.

The Meselson-Stahl Experiment

Design:

1. E. coli were grown for many generations in medium containing heavy nitrogen ($^{15}$N) — all DNA became uniformly "heavy"
2. Cells were transferred to medium containing light nitrogen ($^{14}$N)
3. DNA was extracted after each generation and centrifuged in a CsCl density gradient

Results:

Conclusion: DNA replication is semiconservative — each daughter molecule contains one parental strand and one newly synthesized strand.

Why this rules out other models:

• Conservative would show heavy + light bands at generation 1 (not intermediate)
• Dispersive would show all intermediate bands that become progressively lighter (never a pure light band)
• Only semiconservative predicts one intermediate band at generation 1, then both intermediate and light at generation 2

Checkpoint — Semiconservative Replication

Origins of Replication

DNA replication begins at specific sequences called origins of replication:

Prokaryotes:

• Single circular chromosome with one origin (oriC in E. coli)
• Replication proceeds bidirectionally from the origin, creating two replication forks that move in opposite directions and meet on the opposite side of the chromosome
• Total replication time: ~40 minutes

Eukaryotes:

• Multiple linear chromosomes with many origins (30,000-50,000 in human cells)
• Multiple origins allow the much larger genome to be replicated within the S phase time window
• Each origin fires once per S phase (controlled by the licensing system — pre-replication complexes mark origins for use)
• Adjacent origins define a replicon — the segment of DNA replicated from one origin
• Replication forks from adjacent origins meet and the replicons fuse

Why multiple origins? Human DNA polymerase moves at ~50 nucleotides/second. With 6.4 billion bp and bidirectional replication from one origin, it would take ~2 years. Multiple origins reduce this to ~8 hours.

Key Terms — Replication Model

Exit Ticket

The Replication Machinery

Part 3 of 7

DNA replication requires a team of enzymes and proteins working together at the replication fork — the Y-shaped region where the parental double helix is unwound and new strands are synthesized.

Key Enzymes and Proteins

Why primers? DNA polymerase can only ADD nucleotides to an existing 3'-OH group. It cannot start a new strand from scratch. Primase (an RNA polymerase) CAN start de novo, providing the initial 3'-OH.

Checkpoint — Replication Enzymes

Leading and Lagging Strands

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

Leading strand:

• Template runs 3' → 5' (so new strand grows 5' → 3' toward the fork)
• Synthesized continuously with a single primer
• DNA polymerase moves in the same direction as the fork

Lagging strand:

• Template runs 5' → 3' (so new strand must grow 5' → 3' AWAY from the fork)
• Synthesized discontinuously as a series of short fragments called Okazaki fragments
  • ~1000-2000 nt in prokaryotes; ~100-200 nt in eukaryotes
• Each Okazaki fragment requires its own RNA primer
• After synthesis, DNA Polymerase I removes the RNA primers and fills in with DNA
• DNA Ligase seals the remaining nicks, joining the Okazaki fragments into a continuous strand

The lagging strand is the "complex" strand — it requires repeated cycles of priming, extension, primer removal, gap filling, and ligation. This makes lagging strand synthesis more error-prone and slower than leading strand synthesis.

Checkpoint — Leading and Lagging

Key Terms — Replication Machinery

Exit Ticket

Proofreading and DNA Repair

Part 4 of 7

DNA replication must be extraordinarily accurate — the error rate is approximately 1 mistake per 10$^{10}$ base pairs in E. coli. This remarkable fidelity is achieved through three layers of error correction.

Three Layers of Replication Fidelity

Layer 1: Base selection by DNA polymerase (~10$^5$ accuracy)

• DNA polymerase has a tight active site that favors correct Watson-Crick base pairs (A-T, G-C)
• Incorrect bases fit poorly and are rejected before incorporation
• Error rate: ~1 in 100,000

Layer 2: Proofreading (3' → 5' exonuclease activity, ~10$^2$ improvement)

• DNA polymerase has a built-in editor: if a wrong nucleotide is incorporated, the polymerase detects the mismatch (distortion in the helix)
• The polymerase reverses direction and removes the incorrect nucleotide using 3' → 5' exonuclease activity
• A correct nucleotide is then inserted
• Combined error rate: ~1 in 10$^7$

Layer 3: Mismatch repair (MMR, ~10$^3$ improvement)

• After replication, mismatch repair proteins scan the newly synthesized DNA
• They detect and correct remaining mismatches
• The key challenge: distinguishing which strand has the error (old vs. new strand)
  • In E. coli: the parental strand is methylated (GATC sites); the new strand is not yet methylated, so repair enzymes know to fix the new strand
  • In eukaryotes: the new strand is identified by the presence of nicks (gaps not yet sealed)
• Combined final error rate: ~1 in 10$^{10}$

Checkpoint — Proofreading and Repair

DNA Damage and Additional Repair Mechanisms

Beyond replication errors, DNA is constantly damaged by environmental and metabolic factors:

Types of DNA damage:

• Deamination: Spontaneous loss of an amino group from cytosine, converting it to uracil (if not repaired, G-C becomes A-T after replication)
• Depurination: Loss of a purine base (A or G) from the sugar-phosphate backbone (~5000 per cell per day)
• Thymine dimers: UV light causes adjacent thymines to form covalent bonds (pyrimidine dimers), distorting the helix
• Oxidative damage: Reactive oxygen species (ROS) modify bases (e.g., 8-oxoguanine mispairs with adenine)
• Alkylation: Chemical agents add alkyl groups to bases

Repair pathways:

Xeroderma pigmentosum (XP): A genetic disease caused by mutations in NER genes. Patients cannot repair UV-induced thymine dimers and are extremely sensitive to sunlight, with high rates of skin cancer.

Checkpoint — DNA Repair

Key Terms — DNA Repair

Exit Ticket

Telomeres and the End Replication Problem

Part 5 of 7

Linear chromosomes in eukaryotes face a unique challenge: the end replication problem. This problem does not exist in prokaryotes because their chromosomes are circular.

The End Replication Problem

The problem:

• On the lagging strand, an RNA primer must initiate each Okazaki fragment
• At the very end of the chromosome (3' end of the template), the last RNA primer is synthesized, and DNA polymerase extends from it
• When this primer is removed, there is a short gap at the 5' end of the new strand that CANNOT be filled — there is no upstream 3'-OH for DNA polymerase to extend from
• Result: the daughter strand is slightly shorter than the parent

Consequence: With each round of replication, chromosomes get shorter at both ends. After many divisions, essential genes near the ends would be lost.

Telomeres — The Protective Solution

Telomeres are repetitive, non-coding DNA sequences at the ends of linear chromosomes:

• Human telomere repeat: TTAGGG (repeated 1000-2000 times, totaling 5-15 kb)
• Telomeres provide a "buffer zone" of expendable sequence — shortening removes repeats, not genes
• Telomeres also form a protective structure called a T-loop (the 3' overhang folds back and invades the double-stranded region) with a protein complex called shelterin that prevents the cell from recognizing chromosome ends as DNA breaks

Hayflick Limit: Normal somatic cells can divide approximately 50-70 times before telomeres become critically short. At this point, cells enter replicative senescence (a permanent G$_0$ state) or undergo apoptosis. This is a tumor-suppression mechanism.

Checkpoint

Telomerase — Extending the Ends

Telomerase is a specialized enzyme that extends telomeres, counteracting the end replication problem:

Structure:

• Telomerase is a ribonucleoprotein (protein + RNA)
• Contains TERT (telomerase reverse transcriptase) — the catalytic protein subunit
• Contains TERC (telomerase RNA component) — includes a template sequence complementary to the telomeric repeat

Mechanism:

1. The TERC template (3'-AAUCCC-5') base-pairs with the 3' overhang of the telomere
2. TERT extends the 3' end using the RNA template (reverse transcription — RNA → DNA)
3. Telomerase translocates and repeats, adding multiple TTAGGG repeats
4. Primase then synthesizes a primer on the extended 3' overhang
5. DNA polymerase fills in the complementary strand
6. The primer is removed, leaving a slightly extended chromosome

Telomerase expression:

• Active in: germ cells, stem cells, early embryonic cells — these cells must divide indefinitely
• Inactive in: most somatic cells — contributes to the Hayflick limit and aging
• Reactivated in: ~85-90% of cancers — telomerase reactivation grants immortality

Cancer Connection: Telomerase reactivation is one of the hallmarks of cancer. Drugs targeting telomerase (e.g., imetelstat) are being developed as potential cancer therapies. However, targeting telomerase could also affect stem cells, posing a therapeutic challenge.

Checkpoint — Telomerase

Key Terms — Telomeres

Exit Ticket

Problem-Solving Workshop — DNA Replication

Part 6 of 7

This workshop applies DNA replication concepts to experimental scenarios and quantitative problems.

Scenario 1: Replication Fork Analysis

A researcher treats E. coli with radioactive thymidine ($^3$H-thymidine) for a brief pulse, then chases with unlabeled thymidine. After autoradiography of the replicating DNA:

• The label appears as a band along the newly synthesized DNA
• The leading strand shows a continuous band of label
• The lagging strand shows a series of short labeled segments (Okazaki fragments) with gaps where primers were located

If the pulse is very short (seconds), only the most recently synthesized DNA is labeled. The leading strand shows label near the fork, while the lagging strand shows label in the most recently completed Okazaki fragment.

If the chase is long enough, DNA Pol I replaces primers with DNA and ligase joins fragments, so the lagging strand eventually looks continuous.

Scenario 1 Questions

Scenario 2: Density Gradient Predictions

Starting with one double-stranded DNA molecule where BOTH strands are labeled with $^{15}$N (heavy):

After 1 generation in $^{14}$N:

• 2 molecules, each with one $^{15}$N strand + one $^{14}$N strand = 2 intermediate

After 2 generations in $^{14}$N:

• 4 molecules total
• 2 have one $^{15}$N + one $^{14}$N = 2 intermediate
• 2 have both $^{14}$N = 2 light

After n generations:

• Total molecules = $2^n$
• Intermediate molecules = always 2 (the two original parental strands + a new partner)
• Light molecules = $2^n - 2$
• Heavy molecules = 0 (after generation 1)

Quantitative AP Tip: The number of intermediate-density molecules never changes (always 2) because the two original heavy strands are conserved indefinitely, each paired with a new light strand.

Scenario 2 Questions

Apply Your Knowledge

Exit Ticket — Workshop

AP Review — DNA Replication

Part 7 of 7

Comprehensive AP-exam-style questions integrating all DNA replication concepts.

Key Principles Summary

1. DNA replication is semiconservative — each daughter molecule has one parental and one new strand (Meselson-Stahl)
2. Replication is bidirectional from origins and proceeds in the 5' → 3' direction only
3. The leading strand is continuous; the lagging strand is discontinuous (Okazaki fragments)
4. Primase provides RNA primers; DNA Polymerase III extends; Pol I removes primers; Ligase seals nicks
5. Three layers of fidelity: base selection, proofreading (3'→5' exonuclease), and mismatch repair
6. Multiple DNA repair pathways (BER, NER, HR, NHEJ) protect against different types of damage
7. Telomeres protect chromosome ends; telomerase counteracts the end replication problem

AP-Style Questions — Set 1

AP-Style Questions — Set 2

Comprehensive Matching

Final Review

Final Exit Ticket