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16 Cards in this Set

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Summarize Replication in Prokaryotes
<img src="pastepiwbxs.jpg" />
Compare and contrast DNA Polymerase I vs DNA Polymerase III in E. Coli
DNA Poly I removes RNA primers, fills in nucleotides and repairs DNA. It is made of polA and can 5' to 3' exonuclease, which Poly III can't.
DNA Poly III is the main polymerase in bacteria. It is bigger, faster and made of polC. It is made of 3 subunits - the alpha subunit with catalyses phophodiester bond formation, an epsilon subunit which performs 3' to 5' proof-reading exonuclease activity and a theta subunit which has no catalytic activity.
Both can have conditionally lethal mutations, do 5' to 3' polymerisation and 3' to 5' exonuclease ability.
Explain the significance of the origin of replication
This is the spot where the helicase starts to unzip the DNA. It forms a replication bubble in prokaryotes where replication heads out in opposite directions.
You have to have a code to tell the helicase where to start. In E. coli, those are tandem 13-mer sequences followed by four 9-mer initiation protein binding sites.
Explain the problems with unwinding the DNA double helix
Supercoiling
Positive supercoils tighten the DNA and stop helicase from unzipping it.
Negative supercoils loosen DNA and make it easier to unzip.
Describe the role of helicase enzymes in unwinding DNA prior to its replication
Helicase has to unwind the DNA to replicate it
Describe the role of single-strand binding protein in DNA replication
SSB Protein (prokaryotes) or Replication Protein A - RPA (eukaryotes) keep DNA from reannealing once it has been unzipped
Describe the role of Topoisomerases in DNA replication
Topoisomerases relax supercoiling as helicase unwinds the DNA. This keeps it from becoming too tight to unzip.
Prokaryotes
Topo I only relaxes negative supercoils. DNA independent.
Topo II is called DNA Gyrase in prokaryotes. DNA Gyrase can resolve positive and negative supercoils as well as create both types supercoils. Also needed to seperate the two daughter chromosomes after replication.
Eukaryotes
Topo I relaxes positive and negative supercoils, unlike Pro Topo I. DNA independent.
Topo II cuts both strands of ds DNA, another duplex DNA is passed through the cut ends and the break is resealed. It relaxes both positive and negative supercoils and is needed to decatenate interlinked molecules. Can only create positive supercoils.
Outline the order of events from RNA priming to completed DNA during the replication in E. coli
1. RNA primase synthesises a short primer.
2. DNA polymerase recognises the primer and synthesizes the new DNA chain.
3. DNA polymerase associates with a sliding clamp to increase its processivity.
4. RNA primer is removed by the action of a ribonuclease, gap is filled in by DNA poly II and two adjacent fragments are ligated.
Leading Strand - It just starts and doesn't stop
Lagging Strand - RNA Primer is elongated by DNA Polymerase III until another stretch of RNA if encountered
RNA primer is cut out by DNA Polymerase I, one ribonucleotide at a time
The gap is filled by DNA Polymerase I
Remaining nick is sealed by DNA Ligase
Eukaryotic DNA Polymerases
<img src="pastebmta94.jpg" />
Differentiate between DNA replication in eukaryotes and prokaryotes
<img src="pastemz_kar.jpg" />
Topotecan
Action: poison eukaryotic Topo I
Mechanism: Stabilizes the enzyme cleavage complex Use: Cancer treatment
Name Brands: Camptothecin, Topotecan, Etoposide
Actinomycin D
Action: Prevents the formation of regions of single-stranded DNA. Inhibits the action of topo II, preventing the removal of supercoils. Inhibits RNA transcription
Mechanism: Large ring intercalates between adjacent guanine-cytosine bases, filling the minor groove
Use: Cancer treatment, antibiotic
Name Brand: Dactinomycin
Describe the role of telimeres and telomerase in eukaryotic DNA replication
Telomeres have thousands of repeats of G-rich nucleotides and once they are eaten away you can no longer replicate that DNA. The 3' ends of the chromosome extend 18 nucleotides beyond the 5' ends and then binds back on itself preventing Recombination and the ends being recognized as damage. Telomerase binds to the 3' end of the DNA and elongates in the 5' to 3' direction. Once the telomere is elongated, the lagging strand can be completed by DNA polymerase. Telomerase is supressed in adults but cancer cells avoid this.
Cytosine arabinoside (araC)
Action: Ribose sugar is replaced by arabinose, acts as substrate for several human DNA polymerases
Mechanism: While it has a 3' OH for further chain elongation, it's 2' OH is bent upwards, causing steric hinderance and chain termination.
Use: Cancer
Quinolone
Action: Stabilizes Topo II cleavage complex keeping them from reforming<br />Use: Antibiotic. Only affects DNA Gyrase