DNA Replication Lab Names:_____________________________
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In this lab we will investigate the process of DNA replication. DNA is a double helix(twisted ladder) nucleotide polymer. A nucleotide consists of one nitrogenous base(Adenine, Cytosine, Guanine, or Thiamine), one deoxyribose sugar and a phosphate group. Each nucleotide has one 3’ end with an exposed hydroxyl(-OH) group and one 5’ with an exposed phosphate. The 3’ end can connect to the 5’ end by means of a phosphodiester bond but 3’ cannot connect to 3’ and 5’ cannot connect to 5’.
Phosphodiester bond
Hydrogen bonds
DNA is double stranded and the strands are linked by hydrogen bonds between the different bases. This linking follows Chargaff’s rules…
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The proportion of A always equals that of T and the proportion of G always equals that of C.
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It follows that there is always an equal proportion of purines(A and G) and pyrimidines(C and T)
Each pyrimidine links to one of the purines.(A to T and G to C respectively)
Because of this using a single strand as a blueprint an identical copy can be produced of the original double stranded DNA. To begin the experiment lay out the cloth. The longest strand will be your leading strand. The other strands make up your lagging strand. Snap the lagging strand together from the pieces so that all the portions of the lagging strand are complimentary to the leading strand.
Once the DNA strands are matched twist the two pieces of cloth together so that they are spiraled.
Why is it necessary that each base to be able to make hydrogen bonds with one and only one other type of base?
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When DNA replication begins initiator proteins bind to the replication origin. A replication origin is a specific sequence of nucleotides. A given DNA strand can have one or multiple replication origins and DNA replication can proceed at multiple replication origins simultaneously. They prime the double stranded DNA to begin to unwind.
Once initiated helicases bind to one strand and move along it pulling the two strands apart. To simulate this take your twisted DNA strand. Stick your finger between the two twisted strands to pull them apart making two single strands. Keep the strands together on one end. The other end should have the 5’ exposed on the lagging strand and
3’ exposed on the leading strand.(it should look like a V at this point)

DNA Replication Lab Names:_____________________________
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The single strands once separated by the helicase are stabilized by single-strand binding proteins . The purpose of these proteins is to prevent rewinding of the DNA strands and from being cleaved.
How might DNA replication be effected if there is a problem with the single-strand binding proteins?
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Replication takes place at an incredible rate of speed. 1000 nucleotides are added per second. To keep up with this rate of replication the parent strands must rotate at 100 revolutions per second. This rate of turning creates a great deal of torque that would tear the strand apart without the presence of Gyrases . Gyrases cleave a strand allowing it to swivel around without the excessive torque and then reseal the strand after the rotation.
The protein that adds bases to the two unwound strands is called DNA
Polymerase III.
DNA Polymerase III 3 subunits
  subunit catalyses the addition of DNA in a 5’ to 3’ direction
 
2
dimer subunit clamps the DNA into the Polymerase
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subunit proofreads in the 3’ to 5’ direction
DNA Polymerase III requires a 3’ end to begin replication. The necessary primer is made of about ten RNA nucleotides. These are similar to the DNA nucleotides except they have a ribose sugar instead of a deoxyribose sugar. (In this lab they will look identical to DNA nucleotides except have an R printed on them.) These RNA nucleotides are added by a RNA polymerase called primase . RNA is used instead of DNA because the first segment of added nucleotides are prone to error and this makes it easier to correct later.
To simulate this add one(instead of about 10 in reality) complimentary RNA nucleotide to the exposed end of the leading strand. It should look something like this…
After a primer is added the replication proceeds with DNA nucleotides being added by DNA polymerase III in a 5’ to 3’ direction(toward the replication fork)
Why is a primer RNA necessary for the replication?
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DNA Replication Lab Names:_____________________________
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Replication in the lagging strand is a bit different. DNA polymerase III must proceed in a 5’ to 3’ direction, to do this the lagging strand is broken into chunks called okazaki fragments . These fragments are about 100 to 200 nucleotides long in eukaryotes. To create these fragments unsnap the snaps on the lagging strand.
Just like with the leading strand a 5’ RNA primer must be exposed for DNA polymerase III to attach to. Attach a primer to each of the segments just as before. This time however the replication will proceed away from the replication fork. After putting on the primer attach complimentary DNA nucleotides to the strand
DNA replication is semi-discontinuous on the lagging strand. Why would the cell bother breaking the lagging strand apart?
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The temporary RNA primers now must be removed from both the lagging and leading strands. The protein DNA Polymerase I accomplishes this task by replacing the
RNA nucleotides with DNA nucleotides. Replace any RNA nucleotides in your strands with DNA nucleotides. DNA polymerase I also fills in some of the gaps between the different okazaki fragments.
The now replicated okazaki fragments must be reattached to one another. The protein DNA ligase attaches the fragments back together. To simulate this snap the lagging strand back together. The result of this are two strands with identical DNA.
What function does DNA Polymerase I serve in DNA replication?
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How many stands of DNA are the result of replication? Where do these strands come from? Is the replication conservative or semi-conservative?
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What would happen if there was a mutation that stopped DNA ligase from functioning?
Would the cell be able to reproduce DNA successfully?
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Would this pose a problem if a mutation in a gene produced an error in one of these proteins that stopped it’s function for the cell? What about for the entire organism?
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