Premed · Premed · Genetics
Lecture 7: DNA Structure and Replication (Advanced)
Genetics
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the key experiments that identified DNA as the genetic material
- Explain the Watson-Crick model of DNA and Chargaff's rules
- Describe the semi-conservative model of DNA replication and the Meselson-Stahl experiment
- Identify the enzymes and proteins involved in prokaryotic and eukaryotic DNA replication
- Explain the mechanisms of leading and lagging strand synthesis
- Compare and contrast replication in prokaryotes and eukaryotes
Lecture Content
I. DNA as the Genetic Material: Key Experiments
The identification of DNA as the genetic material rested on a series of landmark experiments. In 1928, Frederick Griffith demonstrated transformation in Streptococcus pneumoniae: when heat-killed virulent (smooth, S) bacteria were mixed with live avirulent (rough, R) bacteria and injected into mice, the mice died. A "transforming principle" from the dead S cells had converted live R cells into the virulent S form. In 1944, Avery, MacLeod, and McCarty identified this transforming principle as DNA by systematically destroying proteins, RNA, and DNA in S-cell extracts and showing that only DNase treatment abolished transformation.
Definitive confirmation came from the Hershey and Chase experiment in 1952, which used T2 bacteriophage with protein labeled by ^35S and DNA labeled by ^32P. After infection, the ^32P (DNA) entered the bacteria while the ^35S (protein) remained outside, and progeny phage contained ^32P but not ^35S, proving that DNA was the molecule carrying genetic information.
Chargaff's rules, established in 1950, provided a critical clue to DNA structure: the percentage of adenine equals the percentage of thymine, and the percentage of guanine equals the percentage of cytosine (%A = %T and %G = %C). The ratio of A+T to G+C is species-specific. These base-pairing relationships proved consistent with the Watson-Crick model of DNA.
II. Watson-Crick DNA Structure
The double helix model was proposed by Watson and Crick in 1953, drawing on Chargaff's rules and the X-ray diffraction data of Rosalind Franklin and Maurice Wilkins, most notably Franklin's Photo 51. The key features of B-form DNA, the most common form in living cells, include two antiparallel polynucleotide strands wound in a right-handed helix, with the sugar-phosphate backbone on the outside and the bases oriented toward the interior. Complementary base pairing holds the strands together: adenine pairs with thymine through 2 hydrogen bonds (A=T), while guanine pairs with cytosine through 3 hydrogen bonds (G≡C). The helix contains approximately 10 base pairs per turn, spans 3.4 nm per full turn with 0.34 nm between adjacent base pairs, and has a diameter of approximately 2 nm. The major groove and minor groove provide access points through which proteins recognize specific DNA sequences. Other DNA forms include A-form (right-handed, wider and shorter, found in dehydrated DNA and RNA-DNA hybrids) and Z-form (left-handed with a zigzag backbone, found in alternating purine-pyrimidine sequences).
<image>Panel A: The Watson-Crick double helix structure with labeled features — antiparallel strands (5'→3' and 3'→5'), major groove, minor groove, sugar-phosphate backbone, base pairs, dimensions (3.4 nm per turn, 0.34 nm per base pair, 2 nm diameter). Panel B: Detailed hydrogen bonding diagram for A-T base pair (2 H-bonds) and G-C base pair (3 H-bonds), showing the chemical structures of each base with atoms numbered. Panel C: Timeline of key experiments leading to the discovery of DNA structure: Griffith (1928), Avery (1944), Chargaff (1950), Hershey-Chase (1952), Watson-Crick (1953).</image>
III. Semi-Conservative Replication
Three models of DNA replication were initially proposed: the conservative model, in which the parent helix remains intact and an entirely new daughter helix is formed; the semi-conservative model, in which each daughter helix contains one parental strand and one new strand; and the dispersive model, in which parental DNA segments are interspersed with new DNA in both strands.
The Meselson and Stahl experiment of 1958 elegantly proved the semi-conservative model. They grew E. coli in medium containing ^15N (heavy nitrogen), so that all DNA was "heavy." Upon transfer to ^14N (light nitrogen) medium, after one generation all DNA had an intermediate density, ruling out the conservative model. After two generations, equal amounts of intermediate and light DNA were observed, ruling out the dispersive model. The results were consistent only with semi-conservative replication, in which each strand of the parent helix serves as a template for synthesis of a new complementary strand.
IV. Prokaryotic DNA Replication
Replication in E. coli begins at a single origin of replication (oriC), a roughly 245 base pair sequence containing AT-rich regions and DnaA boxes. The DnaA protein binds the DnaA boxes and initiates melting at the AT-rich region, which is less stable because A-T base pairs have only 2 hydrogen bonds. Replication proceeds bidirectionally from oriC, with two replication forks moving in opposite directions.
At the replication fork, several key proteins work in concert. Helicase (DnaB) unwinds the double helix ahead of the fork, moving 5' to 3' on the lagging strand template and loaded by the DnaC protein. Single-strand binding proteins (SSBs) stabilize the separated single strands and prevent them from reannealing or being degraded. Topoisomerase (DNA gyrase) relieves the positive supercoiling that accumulates ahead of the fork by introducing negative supercoils through a mechanism of cutting, passing a strand through, and resealing. Primase (DnaG) synthesizes short RNA primers of approximately 10-12 nucleotides, providing the free 3'-OH group that DNA polymerase requires.
DNA Polymerase III is the primary replicative polymerase. It synthesizes DNA exclusively in the 5' to 3' direction, requires a primer with a free 3'-OH, and possesses 3' to 5' exonuclease activity for proofreading. The holoenzyme consists of a core enzyme plus a sliding clamp (beta clamp) and clamp loader complex, which together confer extraordinary processivity of over 500,000 nucleotides per binding event. The synthesis rate is approximately 1,000 nucleotides per second. DNA Polymerase I removes 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.
V. Leading and Lagging Strand Synthesis
Because DNA polymerase can only synthesize in the 5' to 3' direction, the two strands of the replication fork are handled differently. The leading strand is synthesized continuously in the direction of fork movement, requiring only one primer followed by continuous elongation by Pol III. The lagging strand is synthesized discontinuously as Okazaki fragments (1,000-2,000 nucleotides in prokaryotes). Multiple RNA primers are laid down by primase, and Pol III extends each primer until it reaches the previous fragment. Pol I then removes the RNA primers with its 5' to 3' exonuclease and fills in with DNA, and DNA ligase seals the remaining nick.
The trombone model explains how both strands are replicated efficiently at the same fork. The lagging strand template loops back so that both the leading and lagging strand polymerases can move in the same physical direction, with both Pol III cores functioning as part of the same holoenzyme complex known as the replisome.
<image>Panel A: Detailed diagram of a prokaryotic replication fork showing all major components — helicase unwinding DNA, SSBs coating single strands, topoisomerase ahead of the fork, primase synthesizing RNA primers, Pol III on both leading and lagging strands, Okazaki fragments on the lagging strand, Pol I replacing primers, and ligase sealing nicks. All proteins are labeled with their directions of movement. Panel B: The trombone model showing how the lagging strand template loops to allow both polymerases to travel in the same direction, with sequential steps of Okazaki fragment synthesis. Panel C: Diagram of the Meselson-Stahl experiment showing CsCl density gradient results after 0, 1, and 2 generations, with heavy, hybrid, and light DNA bands labeled.</image>
VI. Eukaryotic DNA Replication
Eukaryotic DNA replication differs from prokaryotic replication in several important ways. Human cells contain approximately 30,000-50,000 origins of replication (one every 30-300 kb), which fire at different times during S phase: euchromatin replicates early while heterochromatin replicates late. The Origin Recognition Complex (ORC) binds origins, and licensing occurs when MCM helicase is loaded during G1 phase and then activated in S phase, ensuring each origin fires exactly once per cell cycle.
The replication rate in eukaryotes is slower (approximately 50 nucleotides per second versus 1,000 in bacteria), and Okazaki fragments are smaller (100-200 nucleotides versus 1,000-2,000). The eukaryotic DNA polymerases differ from their prokaryotic counterparts: Pol alpha/primase initiates replication by synthesizing an RNA primer plus a short DNA extension; Pol epsilon handles leading strand synthesis with high processivity conferred by the PCNA clamp; and Pol delta synthesizes the lagging strand, also using PCNA. PCNA (Proliferating Cell Nuclear Antigen) serves as the sliding clamp analogous to the bacterial beta clamp, and RFC functions as the clamp loader.
A unique challenge in eukaryotic replication is the need for histone management. Nucleosomes must be disassembled ahead of the replication fork and reassembled behind it. Parental histones are distributed semi-conservatively to daughter strands, and new histones are deposited by chaperones such as CAF-1. Telomere replication is handled by telomerase, as discussed in the previous lecture. The regulation system ensures that each origin fires exactly once per cell cycle: licensing occurs in G1 when CDK activity is low, firing occurs in S phase when CDK and DDK activity rise, and high CDK activity prevents re-licensing until the next G1 phase.
<image>Panel A: Comparison table of prokaryotic vs. eukaryotic DNA replication features (number of origins, polymerases involved, Okazaki fragment size, replication speed, clamp proteins). Panel B: Diagram of a eukaryotic replication bubble showing bidirectional fork movement from one origin, with Pol alpha initiating, Pol epsilon on the leading strand, Pol delta on the lagging strand, PCNA clamps, and nucleosome reassembly behind the forks. Panel C: Origin licensing and firing model showing ORC binding, MCM loading in G1, and CDK/DDK-dependent activation in S phase, with the block to re-replication illustrated.</image>
