Links
Objectives
- Explain how the structure of DNA suggests a mechanism for its replication
- Describe the design of the Meselson and Stahl experiment that demonstrated semiconservative replication
- Discuss the process of DNA replication
- Identify the key enzymes involved in replication and their functions
Key points
DNA structure
- each nucleotide is composed of a pentose sugar with a phosphate and a nitrogenous base
- early work showed A in equal amount as T, G equal to C
- X-ray diffraction studies by Franklin and Wilkins gave clues to structure
- Watson and Crick made the major conceptual breakthrough, proposing the double helix
- used physical models made to scale of molecules to predict positions of atoms
- accounted for all observations on DNA to date, including equal quantities of bases
- suggested a mechanism of replication just based on structure
- watch an interview in which James Watson describes the insight leading to the idea of complementary base pairing
Semiconservative Replication
- Meselson and Stahl designed an experiment to test different models of replication using ‘heavy’ N to label the nitrogenous base part of DNA
- grew bacteria in 15N media for many generations, then transferred to 14N
- separated DNA by density gradient centrifugation
- different replication models made distinct, testable predictions:
- Semiconservative replication predicts 1 density of DNA after 1 round, 2 after 2 or more rounds of replication
- Conservative replication predicts 2 densities of DNA after 1 or more rounds of replication
- Dispersive replication predicts 1 density after 1 or more rounds of replication
Features of semiconservative replication
- each strand of the double helix serves as a template to produce a complementary strand (video link)
- each new DNA molecule is made up of one old, one new strand
Overview of Replication
- initiation: proteins open double helix, exposing single strands of DNA to enzymes
- elongation: DNA polymerases add free nucleotide bases in 5′ to 3′ direction
- termination: polymerases fall off template strands
Replication in Prokaryotes in detail
- begins at a specific sequence of DNA known as the origin of replication (oriC), rich in AT sequences
- DNA polymerase enzymes only add free nucleotides to the -OH on the 3′ carbon, thus replication proceeds from 5′ end to 3′ end of polynucleotide
- DNA polymerase III is the key enzyme for replication in prokaryotes
- primase adds a short sequence of RNA ‘primer’ to DNA template
- primase does not require the free 3′-OH group to catalyze addition
- DNA helicases unwind the double helix, require ATP
- once unwound, DNA binds to single-strand binding proteins (SSBs) that prevent re-pairing
- topoisomerases bind to double-stranded DNA and relieve torsional strain
Leading and lagging strands
- leading strand synthesis is toward the replication fork (where DNA is unwinding), can be replicated from a single primer
- lagging strand is away from replication fork, requires repeated priming events, which creates Okazaki fragments
- DNA replication on the lagging strand is discontinuous
- DNA polymerase I replaces RNA primers with DNA
- DNA ligase joins Okazaki fragments by forming phosphodiester bond
Eukaryotic differences
- larger genome of Eukaryotes requires multiple origins, forming distinct replicons
- the overall process is similar but involves more components
- Eukaryotic chromosomes are linear, not circular, presenting a problem for replication on the lagging strand at the end
- telomerase solves this problem by having a built-in RNA template for repeating telomere sequence
- keeps telomeres from shortening after each replication cycle
In-class activities
Questions for Practice
- In what way did the structure of DNA proposed by Watson and Crick contain a mechanism for replication ‘built-in’?
- Describe the experiment that showed DNA replication was semi-conservative.
- What is the role of _________ (helicase, topoisomerase, primase, DNA polymerase III, polymerase I) in DNA replication?
- What is an Okazaki fragment?