Premed · Premed · General Biology 1
Lecture 20: DNA Structure and Replication
General Biology I — Molecular & Cellular
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the experiments that established DNA as the genetic material
- Explain the Watson-Crick model of DNA structure, including base pairing and antiparallel orientation
- Describe the semiconservative model of DNA replication and the evidence supporting it
- Explain the roles of the major enzymes and proteins involved in DNA replication
- Distinguish between leading and lagging strand synthesis and explain why Okazaki fragments form
Lecture Content
I. Identifying DNA as the Genetic Material
A. Griffith's Experiment (1928)
Frederick Griffith studied two strains of Streptococcus pneumoniae: the virulent S (smooth) strain, which possessed a polysaccharide capsule, and the non-virulent R (rough) strain, which lacked one. When he injected mice with heat-killed S bacteria mixed with live R bacteria, the mice died, and live S bacteria were recovered from their blood. Griffith concluded that some "transforming principle" from the dead S cells had converted R cells into virulent S cells, but he did not identify the chemical nature of this principle.
B. Avery, MacLeod, and McCarty (1944)
This team systematically purified the transforming principle and tested its identity by selectively destroying each class of macromolecule. Destroying proteins or RNA had no effect on transformation, but treating the extract with DNase (which degrades DNA) eliminated transforming ability entirely. They concluded that DNA is the genetic material--though many scientists, convinced that the greater chemical complexity of proteins made them the more likely candidate, remained skeptical.
C. Hershey-Chase Experiment (1952)
Alfred Hershey and Martha Chase used bacteriophage T2 to settle the question definitively. They labeled phage DNA with radioactive 32P and phage protein with radioactive 35S, then allowed the phages to infect E. coli. After infection, 32P was found inside the bacteria while 35S remained outside, confirming that DNA, not protein, is the genetic material injected into the host cell.
D. Chargaff's Rules (1950)
Erwin Chargaff analyzed the base composition of DNA from various organisms and discovered two key regularities: the amount of adenine always equals the amount of thymine (A = T), and the amount of guanine always equals the amount of cytosine (G = C). The ratio of purines to pyrimidines is therefore always 1:1, though the overall base composition varies between species. These rules provided essential clues for deciphering DNA's three-dimensional structure.
II. The Structure of DNA
In 1953, James Watson and Francis Crick proposed the double helix model of DNA, drawing on Chargaff's base-pairing rules, X-ray crystallography data from Rosalind Franklin and Maurice Wilkins (particularly the famous Photo 51), and physical model building. The structure they described has several key features. Two polynucleotide strands wind around each other in a right-handed helix, running in antiparallel directions (one 5' to 3', the other 3' to 5'). The sugar-phosphate backbone, formed by deoxyribose sugars linked by phosphodiester bonds, runs along the outside. The nitrogenous bases are stacked on the inside, perpendicular to the helix axis, and held together by complementary base pairing: adenine pairs with thymine through two hydrogen bonds, and guanine pairs with cytosine through three. Because a purine always pairs with a pyrimidine, the helix maintains a consistent diameter of 2 nm. The helical twist creates major and minor grooves on the surface, which are important sites of protein-DNA interaction. Each complete turn of the helix spans 3.4 nm and encompasses 10 base pairs.
<image>A detailed diagram of the DNA double helix. Left: A space-filling model of the overall helix structure showing the major and minor grooves, the sugar-phosphate backbone, and the 2 nm diameter. Right: An unrolled "ladder" view showing the antiparallel strands with 5' and 3' ends labeled. Base pairs are shown in the middle: A-T connected by 2 hydrogen bonds (dashed lines) and G-C connected by 3 hydrogen bonds. The phosphodiester bonds connecting nucleotides in the backbone are labeled. Dimensions are annotated: 3.4 nm per turn, 0.34 nm between base pairs, 2 nm width.</image>
III. DNA Replication — Overview
DNA replication is semiconservative: each daughter double helix consists of one original (parental) strand and one newly synthesized strand. This was elegantly demonstrated by the Meselson-Stahl experiment (1958), in which E. coli were grown in heavy nitrogen (15N) medium and then transferred to light nitrogen (14N) medium. After one generation, all DNA had an intermediate density (one heavy strand plus one light strand). After two generations, half the DNA was intermediate and half was light. This result was consistent only with semiconservative replication, ruling out the conservative and dispersive models.
Several fundamental principles govern replication. It is bidirectional, proceeding in both directions from each origin. New strands are always synthesized in the 5' to 3' direction, because DNA polymerase can only add nucleotides to a free 3'-OH group. The substrates are deoxyribonucleoside triphosphates (dNTPs), and the release and hydrolysis of pyrophosphate provides the thermodynamic driving force.
IV. Steps of DNA Replication (in E. coli)
A. Initiation
Replication begins at origins of replication--specific DNA sequences designated oriC in E. coli (a single origin) or tens of thousands of origins in eukaryotes. The DnaA protein binds to oriC and pries apart the two strands, beginning at AT-rich regions (which are easier to melt because A-T base pairs have only two hydrogen bonds). Helicase (DnaB) unwinds the double helix at the replication fork, powered by ATP hydrolysis. Single-strand binding proteins (SSBs) coat the exposed single strands, preventing them from re-annealing or being degraded by nucleases. Topoisomerase (gyrase) relieves the supercoiling tension that builds up ahead of the advancing replication fork by cutting, rotating, and re-sealing the DNA.
B. Elongation
Because DNA polymerase cannot initiate synthesis from scratch, primase (DnaG) first synthesizes a short RNA primer (~10 nucleotides) complementary to the template strand, providing the free 3'-OH that polymerase requires. DNA Polymerase III, the main replicative enzyme in E. coli, then extends the primer by adding dNTPs complementary to the template in the 5' to 3' direction. Held in place by the sliding clamp (beta clamp), Pol III achieves high processivity and incorporates nucleotides with remarkable fidelity, aided by its built-in 3' to 5' exonuclease (proofreading) activity that detects and removes mismatched bases, reducing the error rate to approximately 1 per 10^7 bases.
Because the two template strands run antiparallel and DNA polymerase synthesizes only 5' to 3', the two new strands are made differently. The leading strand is synthesized continuously toward the replication fork, requiring only a single RNA primer. The lagging strand is synthesized discontinuously in short segments called Okazaki fragments (1,000-2,000 nucleotides in E. coli, 100-200 in eukaryotes), each requiring its own RNA primer. These fragments are synthesized in the 5' to 3' direction, away from the replication fork.
DNA Polymerase I removes the RNA primers using its 5' to 3' exonuclease activity and fills the resulting gaps with DNA. DNA ligase then seals the remaining nicks by forming phosphodiester bonds between adjacent Okazaki fragments, producing a continuous daughter strand.
<image>A detailed diagram of a replication fork. The parental DNA double helix is shown being unwound by helicase at the fork. Topoisomerase is ahead of the fork relieving supercoiling. SSBs coat the single-stranded regions. On the leading strand (top), DNA Pol III synthesizes continuously toward the fork with one primer at the origin. On the lagging strand (bottom), primase lays down short RNA primers (shown in red), DNA Pol III extends each primer to form Okazaki fragments, DNA Pol I removes primers and fills gaps, and DNA ligase seals the nicks. The sliding clamp is shown encircling the DNA at each polymerase. Arrows indicate the 5' to 3' direction of synthesis on both strands. The overall direction of fork movement is indicated.</image>
V. Eukaryotic DNA Replication — Key Differences
Eukaryotic genomes are vastly larger than bacterial genomes and require multiple origins of replication--the human genome has approximately 30,000 to 50,000 origins. These are licensed in G1 phase by the origin recognition complex (ORC), Cdc6, Cdt1, and the MCM helicase, and are fired (activated) during S phase. Eukaryotic cells employ distinct DNA polymerases for different tasks: Pol alpha/primase synthesizes the initial RNA primer and a short DNA extension, Pol epsilon replicates the leading strand, and Pol delta handles the lagging strand. The eukaryotic sliding clamp is called PCNA. A unique complication arises from the packaging of DNA with histones: chromatin must be disassembled ahead of the replication fork and reassembled behind it, requiring coordinated histone chaperone activity.
VI. The End Replication Problem and Telomeres
Linear chromosomes pose a challenge that circular bacterial chromosomes avoid: the end replication problem. When the final RNA primer on the lagging strand is removed, there is no upstream 3'-OH to fill the gap, resulting in a loss of approximately 50 to 200 base pairs from each chromosome end with every round of replication. Telomeres solve this problem by capping chromosome ends with thousands of repeats of a short sequence (TTAGGG in humans) that acts as a disposable buffer. The shelterin protein complex associates with telomeres and prevents them from being recognized as DNA damage by the repair machinery.
Telomerase is a specialized ribonucleoprotein enzyme (a reverse transcriptase) that extends telomeres by using an internal RNA template to add TTAGGG repeats to the 3' overhang. Telomerase is active in germ cells, stem cells, and--notably--most cancer cells, but it is inactive in the majority of somatic cells. As a result, somatic cell telomeres shorten with each division until they reach a critical length, triggering cellular senescence or apoptosis. The Hayflick limit--approximately 50 to 70 divisions for normal human cells--reflects this progressive telomere erosion. Telomere biology thus sits at the intersection of aging, cancer, and stem cell function.
<image>A diagram of the end replication problem and telomerase action. Top panel: A linear chromosome end showing the leading strand replicated completely to the tip, but the lagging strand having a gap where the last RNA primer is removed. An arrow indicates the progressive shortening with each replication cycle. Middle panel: Telomere structure — the 3' G-rich overhang forming a T-loop that tucks back into the double-stranded region, stabilized by shelterin proteins. Bottom panel: Telomerase extending the 3' overhang — the enzyme (with its integral RNA template shown in red, complementary to TTAGGG) base-pairs with the overhang, adds nucleotides, translocates, and repeats. After extension, primase and DNA polymerase fill in the complementary strand.</image>
