BIO 1140 Lecture Notes - Lecture 20: Dna Ligase, Dna Clamp, Semiconservative Replication

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Thu, March 24-Lecture 20 DNA replication pt 2
Slide 3
Needs to unwind the strands - helicase. In order to prevent them from recoiling, SSBs, evenly spaced, just
so that it is stable enough that it won't recoil. Topoisomerase upstream of either side of the fork to prevent
supercoiling of the DNA that has not yet been unwound (not yet part of the replication bubble).
Slide 4
Primase places the primers.
Slide 5
Primers are RNA. DNA polymerase 3 can only bind to double stranded DNA. To trick polymerase into
thinking there is a gap to fill, primes adds 6-10 nucleotides of DNA. Complementary strand will be a
short RNA sequence followed by a longer DNA sequence.
Slide 7
As it adds the new strand, adding it on the 3rd carbon sugar where the hydroxyl group is, 5th carbon of
the incoming nucleotide. To make sure that DNA polymerase III remains stable and interacting with the
DNA, and make sure it goes all the way to the end of the strand, a protein accompanies it to make sure it
continues its job - a sliding DNA clamp. It doesn't contribute to any of the actions of DNA polymerase
III, only ensuring polymerase continues doing its job and stays clamped on the strand, supports it. *It isn;t
bound to it either.
Slide 9
DNA polymerase only elongates the new strand from 5' to 3', always goes from the origin to the fork.
Slide 10
You should be able to picture where primase will go (know where leading and lagging strands are).
Remove primers, DNA polymerase I will remove the RNA nucleotides and replace them with DNA
nucleotides (different sugar). This will leave a gap? When you exchange the nucleotides, there is no bond
between them, which will create the gap. You need to bond the two nucleotides together- DNA ligase.
Slide 12
After nick is filled by DNA ligase, you end up with the fully replicated daughter strand with each parental
strand. Can see now why semi-conservative replication makes the most sense?
Slide 14
DNA polymerase III does two things: adds complementary daughter strand (5' to 3' activity) AND it
proofreads (3' to 5' exonuclease activity) makes sure the strands are correct. Can backup, remove a
mismatch, and add the correct nucleotide. Like forwards and reversing a car. It proofreads WHILE its
replicates. If it adds the wrong nucleotide, it'll back up and fix it, then continue replicating.
Slide 15
What if polymerase III doesn't fix an error? We have other enzymes, endonucleases that are able to see
mismatches, come in, remove a bit of DNA and adds new nucleotides to correct the mismatch. Can't
remove just the mismatched nucleotide, removes a small section. Leaves a nick, so DNA ligase would
come in and seal the nick. What if the mismatch still isn't corrected? Stays behind, mutations could occur.
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