structure (chapter 10, pages 266 – 278) and replication of dna (chapter 12, pages 318 – 334)
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Structure of DNA
• Designate the Nucleotides – Purines• Guanine = G• Adenine = A
– Pyrimidines• Thymine = T• Cytosine = C
Structure of DNA
• Nucleotides join together, forming a polynucleotide chain, by phosphodiester bonds– The phosphate attached to the 5’ carbon
on one sugar – Attaches to the 3’ hydroxyl (OH) group
on the previous nucleotide
5’-phosphate of last nucleotide chemically bonded to the 3’-hydroxyl of the next-to-last nucleotide
A phosphodiester bond
Structure of DNA
• DNA is a double helix (two strands) held together by hydrogen bonds– Adenine (A) and thymine (T) are paired– Guanine (G) and cytosine (C) are paired– Always a purine pairs with a pyrimidine
The two polynucleotide strands (the backbones) in the double helix run in opposite directions, and are said to be anti-parallel
5’-end
3’-end5’-end (free 5’-phosphate)
3’-end (free 3’-OH)
Because of the pairing (A-T; G-C), one polynucleotide chain is always complementary to the base sequence of the other strand
5’-end
3’-end5’-end (free 5’-phosphate)
3’-end (free 3’-OH)
It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.
J. D. Watson and F. H. C. Crick, 1953
Matthew Meselson and Franklin Stahl, 1958
entirely new AND entirely old DNA moleculespresent
ALL DNA moleculesare made upof both oldand new DNA
entirely new DNA moleculespresent BUT not entirely old DNA molecules
Meselson and Stahl
• Experiment– Grew E. coli in a growth medium
containing only 15N (heavy nitrogen)(note: Normal isotope is 14N lighter nitrogen)
– Did this for many generations so that all of the bacterial DNA would be “heavy”
Meselson and Stahl
• Experiment– Then grew the bacteria with 15N
incorporated in their DNA in medium containing only 14N (would be incorporated into the new DNA)
This way they could differentiate the original DNA from newly incorporated DNA
Meselson and Stahl
• Experiment– At each generation • Isolated the DNA • Looked at the density of the DNA in a CsCl
gradient
Matthew Meselson and Franklin Stahl, 1958
entirely new AND entirely old DNA moleculespresent
ALL DNA moleculesare made upof both oldand new DNA
entirely new DNA moleculespresent BUT not entirely old DNA molecules
Great test question:
Predict what the cesium chloride gradients would look like for conservative and dispersive replication!
Meselson and Stahl showed that the semiconservative pattern of replication is what was found
So the DNA double helix unwinds and each strand acts as a template for replication of the new half
Replication
General features:1. There is a specific site where replication begins (origin)
which must be recognized2. The two strands of DNA must be separated3. The original strand becomes the template for the new
DNA strand4. A primer molecule must be added on which the new
DNA chain can be built5. New nucleotides must be added complementary to the
template strand6. The newly synthesized DNA must be edited and joined
into one continuous molecule
Replication
Origin of replication– Where synthesis of new DNA begins– A specific location with a specific sequence of
nucleotides
In bacteria and viruses one origin of replication
In eukaryotes there can be thousands of replication origins
Origins of replication
Initiator ProteinsRecognizesthe Origin of Replication
Start to denature the DNA so each strand can act as a template
Replication
• DNA is unwound by a helicase– Separates the double helix by breaking
the hydrogen bonds
The separated (single strand DNA) is combined with single-strand binding proteins
•Protects DNA from degradation•Keeps the complementary strands from rejoining
Replication
• As DNA is unwound it will tangle and knot, called supercoiling (from the unwinding of the helix)
The supercoiling must be relaxed (the DNA unknotted)
This is done by a class of enzymes called topisomerases (gyrase)
123 4
1 = initiator proteins2 = single strand binding proteins3 = helicase4 = topoisomerase (gyrase)
5’ triphosphates of the four nucleotides must be present (dATP, dGTP, dTTP, dCTP)
Two of the phosphates are cleaved-off, providing energy to run the reaction
The preexisting single strand of DNA is the template strand
Complementary base to the template strand
Phosphates cleaved to provide energy for the reaction
Nucleotide monophosphates are then joined to the 3’OH group
Replication
• DNA Polymerase–Must have a free 3’-OH group onto
which to add the new nucleotides
– No known DNA polymerase is able to initiate chains; thus, requires a primer to start synthesis
Replication
• DNA Polymerase–Must have a free 3’-OH group onto
which to add the new nucleotides
– No known DNA polymerase is able to initiate chains; thus, requires a primer to start synthesis
Must have a primer (which is an RNA molecule)
The primer is synthesized by the enzyme primase (RNA polymerase)
Replication
• Two DNA polymerase enzymes are necessary for replication in E. coli– DNA polymerase I – DNA polymerase III
• Both have polymerase and exonuclease activities (functions)
Replication
• Polymerase:– Synthesize new DNA in the 5’ 3’ direction
• Exonuclease:– Remove nucleotides from the end of a chain
(proofreading and editing functions)• 5’ 3’ (removes primers)• 3’ 5’ (editing, removes incorrect bases)
Replication
• DNA Polymerase I– Synthesize new DNA in the 5’ 3’ direction
• Only synthesizes short sequences of new DNA
– 3’ 5’ exonuclease activity (proofreading)– 5’ 3’ exonuclease activity (remove primers)
• DNA Polymerase III– Synthesize new DNA in the 5’ 3’ direction
• Synthesizes long sequences of new DNA
– 3’ 5’ exonuclease activity (proofreading)
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