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Lecture 14

BIOB11H3 Lecture Notes - Lecture 14: Okazaki Fragments, Dna Clamp, Dna Replication


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs
Lecture
14

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Lecture 14 DNA replication II: Enzymology, Origins, Organization and Licensing
SPs: Figs 13-12, 13, 14, 15, 19, 20
Vocabulary: helicase/primase/primosome/replisome/Sliding clamp/origin/fiber
autoradiography/licensing
Work by Kornberg (1950’s)
-interested in DNA polymerase and how it works, how many subunits it had, what it did,
etc.
-he purified the protein, he took the dna template, added the raw materials for replication
to occur (i.e. deoxynucleotide triphosphates dNTP’s) and he radioactively one type of the
nucleotides. When the radioactive nucleotide is incorporated into the DNA polymer then
the polymer will also become radioactive. This is evidence that DNA Replication is
occurring and radioactive materials are being incorporated into the chromosome.
-Figure 13-7: Kornberg was able to purify a variety of different templates like double
stranded circles, like single stranded circles, like double stranded DNA with partial single
stranded character.
-He discovered that there are two goals for DNA replication: 1. DNA polymerase needs a 3’
hydroxyl group to get started at a prime site and 2. DNA polymerase has to have a template
in front of it to copy. This is why, in figure 13-7, the templates on the right are better
templates than the ones on the left.
Enzymology at the Replication Fork
-What has to go on in order for replication to take place…
- Firstly, some enzyme activities must be assigned. Starting at the origin of replication,
currently the DNA is double stranded and it has to be single stranded in order for DNA
polymerase to read to template so one of the first things that needs to be done is that the
double helix needs to be unwound by the enzyme helicase.

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-Now, to keep the single stranded pieces from snapping back together by complementary
base pairing, single stranded DNA binding proteins exist and they coat the strands so
that they can’t snap back together.
-DNA polymerase can only start at a primed site, so in order to initiate strands, an enzyme
called primase is needed. It lays down a short RNA primer and provides the 3’ hydroxyl
group.
-Now the DNA with an open end and primed site is ready for DNA polymerase to do the job.
So DNA Polymerase is responsible for chain elongation from the primed site (in ecoli, there
are 3 DNA polymerase types and DNA polymerase III is the main replication enzyme).
-Once DNA polymeraseIII is done its job, the RNA primers that still exist need to be
removed and replaced with DNA. This is done by DNA polymerase I which removes the
ribonucleotides and replaces them with deoxyribonucleotides.
-On the “discontinuing” or “lagging” strand, DNA is synthesized discontinuously as short
Okazaki fragments. These need to be put together so to enzyme called DNA ligase is
responsible for sealing that small section and creating the intact polymer chain.
Enzymes at the Replication Fork: Primase
Figure 13-12:
-Double stranded DNA is seen on the left which is the parental strand that needs to be
unwound
-In the middle, is the replication fork where replication is going to occur from right to left
as the DNA unwinds.
-DNA helicase unwinds the DNA (it is a hexamer of 6 identical subunits). Closely associated
with DNA helicase is the enzyme Primase. Primase and Helicase move along together as
unwinding takes place. Primase lays down the RNA primers (green). The space is about 9-
10 nucleotides in length. The complex of Helicase and Primase is known as Primosome.

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-When you see double stranded molecules, it is important to know which way is which. So
always put in the 5’ and 3’.
Replicons, eyes, bubbles and forks
-A unit of replication is referred to as a replicon.
-In the context of double stranded DNA where there is an origin that exists, there is
unwinding by helicase, then there is bidirectional replication.
-Looking at this through a microscope, you begin to see the helix becomes unwound and
you get something that looks like an eye or a bubble - it is wider than it was before.
-As elongation continues, the eye or bubble gets larger. Each eye is about bidirectional
replications so there are two replication forks.
-The fork is where the replicated DNA is meeting the unreplicated parental DNA. Each eye
consists of two forks that are moving in opposite directions.
The leading and lagging strands are continuous and discontinuous so each fork then also
consist of one leading strand going 5’ to 3’ continuously and one lagging strand that is
synthesized as the short Okazaki pieces in a discontinuous fashion.
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