MBG 2040 Lecture Notes - Lecture 7: Cold Spring Harbor Laboratory, James Watson, Differential Centrifugation
2 May 2018
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DNA Replication
Outline:
1. Basic features of DNA replication in vivo
2. DNA replication in prokaryotes
3. Unique aspects of chromosome replication in eukaryotes
Monozygotic Twins - accurate DNA replication
Standard B-form DNA helix of Watson & Crick
• Basic features:
o Antiparallel strands --> polarity
o Complementary base pairs located in the interior (A-T; G-C)
o Right handed double helix (B form DNA)
• Most common form in cells of living organisms
o 10 base pairs per turn
o 0.34 nm between two stacked bases
o 3.4 nm per helical term
• Watson & Crick: this structure has novel features which are considerable biological interest
o James Dewey Watson was born in 1928 in Chicago
o Earned scholarship to University of Chicago at age 15
o BS: 1947 University of Chicago, Zoology
o PhD: 1950 Indiana University (under Salvador Luria, influence by the 'Phage Group'
established by Luria and Max Delbruck)
o 1950: Postdoc at Copenhagen University - wanted to study genes via biochemistry
o 1951: moves to University of Cambridge (Cavendish Laboratory) - collaboration with Francis
Crick
o 1953: Publication of the double helix structure
o 1962: Nobel prize with Crick and Maurice Wilkins
o 1956-1976: faculty member at Harvard
o 1968: Director of Cold Spring Harbor Laboratory
o 1990-1992: head of genome project at NIH
• Central Dogma of Molecular Biology was first articulated by Francis Crick in 1958
o Gene (DNA) undergoes replication
o Through transcription becomes RNA
o Through translation becomes polypeptide (amino acids)
o The discovery of reverse transcriptase in retroviruses and retro-like viruses (Temin &
Baltimore 1970) defied the central dogma
Basic Features of DNA Replication in Vivo
• "it has not escaped our notice that the specific pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material" - Watson & Crick
• The Watson-Crick double helical DNA model predicted DNA replication by a semi-conservative
mechanism
o Parental strand separate --> synthesis of complementary strands --> two identical DNA
molecules
• But…there were other possibilities and theories:
o Semiconservative
o Conservative
o Dispersive
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• By means of an elegant experiment, Meselson and Stahl proved that the semi-conservative model
was the correct mechanism for DNA replication
o Cesium chloride equilibrium-density gradient centrifugation:
• Prepared 6M CsCl solution and added mixture of DNA containing N15 and N14
• Centrifuged at 50,000 rpm for 48-72hrs
▪ *equilibrium is established between centrifugal force and diffusion
• Hole was punched in centrifuge tube and fractions were collected
▪ --> density gradient: density decreased as position in the centrifuge tube
increased (from bottom to top)
• Density of "heavy" (15N) E. coli DNA > "light" (14N) E. coli DNA
o *see slide
o E. coli cells were grown on 15N for several generations (heavy) **DNA was extracted and
analyzed by CsCl density gradient centrifugation after each step
• Cells were transferred onto medium containing 14N for one generation: 1st generation
progeny DNA is hybrid
• For two generations: 2nd generation progeny DNA is half hybrid and half light
• For three generation: 3rd generation progeny DNA is 1/4 hybrid and 3/4 light
• John Cairns: replication begins at an "origin" (OriC in E. Coli)
o *see slide
o An "AT" rich region (13-mer tandem repeats) that undergoes strand separation
o There is only one origin of DNA replication in circular genome of E. coli
o Four binding sites for the initiator protein DnaA
• DNA replication is 'bi-directional' from the origin of replication
o Two replication "forks" begin at the origin and progress bi-directionally around the E. coli
chromosome
o *see slide
DNA Replication in Prokaryotes:
• DNA replication cannot occur de novo
o Primer with free OH group at 3' end
o Template DNA to specify the sequence of new DNA strand
o Substrates: dNTPs
o Mg2+ (what is it's function??)
o DNA polymerase
o (dNMP)N + dNTP (+ DNA template, DNA dependent DNA polymerase) --> (dNMP)n+1 + Ppi
• Nucleic acid synthesis is a one-way traffic, and follows a 5' to 3' direction only
o It requires the free OH group at the 3' carbon of the preceding nucleotide residue, which
initiates a nucleophilic attack on the inner (alpha) phosphate group of the incoming
nucleotide
• There are 5 types of DNA polymerases in E. coli:
o I & III --> chromosomal DNA replication
• 'replicative polymerases'
• Pol III is main
o II, IV and V --> DNA repair functions
• Replicative polymerases (I and III)
o Pol I: responsible for the removal of the RNA primer and filling the gaps
• Has 5' to 3' polymerase and 5' to 3' exonuclease activity
• Proof reading ability: due to its 3' to 5' exonuclease activity
• Not highly processive; synthesis of short fragments of DNA
o Poly III: main replicative polymerase; highly processive
• Has 5' to 3' polymerase activity
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