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Biol. 2320 - Fall 2000 - In-Class Exercise (due today) In-Class Exercise or "If you find a replication fork in the road,
leave it where you found it"
The problem
(solution due at the end of class today): The replication fork of an
actively replicating DNA duplex moves in a single direction, with daughter
strands synthesized on the separated parental strands. Both daughter
strands, like all DNA, are synthesized in a 5'®3'
orientation. One of the two daughter strands is polymerized into the
replication fork (the leading or continuous strand) while the other is
read out of the replication fork (the lagging or discontinuous strand).
The figure from your text shown on the following page (fig 13.8c) is a
misrepresentation. It shows one DNA polymerase enzyme directing continuous
strand synthesis and another separate DNA polymerase directing
discontinuous strand synthesis. In fact, DNA polymerase is a dimer (i.e.
two enzymes hooked together) made up of one monomer that directs
continuous strand synthesis and a slightly different monomer that directs
discontinuous strand synthesis. The dimer can only move in the direction
of the replication fork. How can one enzyme dimer, moving in the direction
of the replication fork, synthesize both the continuous and discontinuous
strands? The enzyme cannot simply stretch. Use the model replication kit
provided to come up with a mechanism for how discontinuous strand
synthesis might work (we know how continuous strand synthesis works). Replication Kit: Double sphere:
DNA polymerase asymmetric dimer Rules: Synthesize the new discontinuous strand from one end
of the strand to the other using the Okazaki fragments (again, ignore
leading strand synthesis for now). You can only synthesize in a 5’®3'
direction; therefore the parent strand must be read in a 3'®5'
(plugs define 5' ends of parental strands). Position the enzyme dimer right at the replication fork (unclip the strands as you go). You can only add an Okazaki fragment when the
appropriate portion of the strand is in contact with the enzyme dimer at
the replication fork. Hints: DNA is not a rigid molecule, particularly when in single stranded form. It is much more flexible, like the model tubing we have used. Do not let your preconceived notions of DNA structure dictate what you consider to be possible. [5 pts] Turn
in one copy of this today: Group members: ____________________________ ____________________________
Describe the solution you came up with in no more
than two sentences - you can also use this space to draw what you did: Thought question: 1.
[2 points] We just had to go through contortions to generate a
workable model that describes the three dimensional structure of the
replication fork. This is all because the DNA polymerase is a dimer and
not two types of free monomer. Provide a biologically relevant reason why
cells might need to keep the two sites of polymerization (the enzymes)
together. Explain in no more than 2 sentences. 2.
[3 points] You just clipped together a whole series of Okazaki
fragments, discontinuously synthesized from the lagging strand. When
Meselson and Stahl performed their experiment that confirned that DNA was
replicated semi-conservatively, they used density gradient centrifugation
to separate different types of DNA. Why didn't they observe Okazaki
fragments? |