MBB 201 Lecture Notes - Lecture 11: Amine, Topoisomerase, Depurination
Chapter 6: DNA Replication, Repair and Recombination
DNA Replication
Models of Replication
• Conservative- parent helix is entirely conserved, and new daughter helix is produced
• Semiconservative- one strand is parent and one is daughter of each helix produced
• Dispersive- in both strands produced there is a mixture of new and old nucleotides in
both
Meselson-Stahl Experiment
• Heavy and light nitrogen nucleotides were placed in a cesium chloride solution
• DNA was allowed to undergo replication
• Both strands formed were the same weight
o Ruled out conservative model
• Results were heated to break the hydrogen bonds holding strands together and then put
in a centrifuge to separate their weights
o One strand was heavier than the other strand
o Ruled out dispersive
• Established the semiconservative model was correct
Template
• Because of base pairing each strand of DNA can be used for replication
• Two new identical helices are produced
• Each daughter ends up with one of the parent strands
o Semiconservative
Beginning of replication
• Initiator proteins binds to specific DNA sequences called replication origins
• Two strands of DNA are opened, hydrogen bonds broken
o This doesn’t require a lot of energy because individual hydrogen bonds are very
weak
• Replication origins are usually rich in A-T because it is easy to pull apart (only two
hydrogen bonds)
• Y shaped junctions at either side of the replication bubble are called replication forks
o They move away from the origin
o Bidirectional replication
Continuous Replication
• DNA polymerase catalyzes the addition of nucleotides to the 3’ end of a growing DNA
strand using one of the parental strands as a template
o Polymerization reaction involves the formation of a phosphodiester bond
between the 3’end of the new DNA and the 5’ of incoming phosphate group
o Energy provided by the hydrolysis of one of the phosphate bonds on incoming
nucleotide
• DNA polymerase does not detach after each monomer is added, it stays clamped to the
DNA
• Strand made in the 5’-3’ direction is the leading strand
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Discontinuous Replication
• DNA that appears to grow in the 3’ to 5’ direction
o DNA polymerase only works in the 5’ to 3’ direction
• DNA is synthesized from small fragments- Okazaki Fragments
• These strands are joined together to form the new strand
o This strand is called the lagging strand because there is a delay in its synthesis
DNA Polymerase
• Only works in the 5’ to 3’ direction
• Makes one error in ever 10^7 nucleotide pairs copied
• Carefully monitors base pairing between incoming nucleotide and template strand Will
only catalyze reaction if pair is correct
• Proofreading: before next nucleotide is added previous one is checked to make sure it is
correct
RNA Primer
• A short length of RNA is synthesized (about 10 bases long) from the template strand and
is used as the starting point for DNA polymerase to continue synthesizing new bases
• Enzyme that synthesizes the RNA primer is primase
o An example of RNA polymerase
• RNA primer is only needed once for the leading strand but is needed many times for the
lagging strand to keep replication going
o Ozaki fragments are elongated until the next primer is reached
• Removing the primer and joining the fragments
o Nuclease degrades the RNA primer
o Repair polymerase (a DNA polymerase) replaces RNA with DNA using end of
adjacent Okazaki fragments as a primer
o DNA ligase joins 5’ phosphate to 3’ hydroxyl of next fragment
• Primers usually contain mistakes as they are not proof read
Proteins part of Replication Machine
• DNA helicase
o Along with single stranded DNA binding proteins
▪ Prevents DNA from closing back up
o Opens or unzips the DNA double helix
o Uses the energy of ATP hydrolysis to propel forwards
o Creates tension in the fork on the other side
• DNA topoisomerase
o Relieves the tension on the forks
o Produce nicks in the backbone to temporarily relieve pressure
o Reseal nicks before falling off DNA
• Sliding clamp
o Keeps DNA polymerase firmly attached to the template
• Clamp loader
o Assembles sliding clamp around DNA using the hydrolysis of DNA
o Required many times on the lagging strand because the DNA polymerase falls
off after each Okazaki fragment
Telomerase
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