MBB 201 Lecture Notes - Lecture 11: Amine, Topoisomerase, Depurination

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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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