Term
| Watson and Crick Model of Replication |
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Definition
The parental strands separate and each serves as a template for aligning new nucleotides for the daughter strands.
Problem: General Not Specific. |
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Term
| Discovering DNA Polymerase |
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Definition
Incubate
a)Template DNA
b)Radioactively labelled nucleotides
c)Cell Enzymes
Observations:
1)Pure Double-Stranded DNA does NOT work (no access point).
2)Single-Stranded template does NOT work (DNA polymerase requires a primer).
3)DNA with gaps DOES allow nucleotide addition. DNA polymerase adds to 3' end of existing strands. |
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Term
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Definition
1)If only simple nucleotides (base + sugar + phosphate) are added to the reaction mixture no DNA synthesis occurs.
2) In order to serve as precursors for DNA synthesis nucleotides must be present in an activated state, as nucleoside triphosphates (made with ATP). |
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Term
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Definition
| The molecule actually incorporated into a growing DNA strand is a nucleoside MONOphosphate (dAMP, dCMP, dGMP, or dTMP). During the act of incorporation, two phosphates (a pyrophosphate) are removed from each dNTP precursor, providing the energy for the polymerization reaction. |
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Term
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Definition
1)DNA Polymerase positions itself at the
end of the growing strand—which has an
exposed C–3 OH group
2)The Polymerase active site lies directly under the 3–OH group.
3)An active site amino acid takes a proton (H+) from the C–3 OH group, now creating a nucleophilic oxygen which subsequently attacks the gamma phosphate, releasing a pyrophosphate.
4) Note that binding places the innermost phosphorous adjacent to the
C–3 OH group.
--SIDE NOTE:The subsequent cleavage of the displaced pyrophosphate, with its high-energy anhydride bond, generates two molecules of low-energy inorganic phosphate(pyrophosphate)—making the DNA polymerisation reaction irreversible. |
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Term
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Definition
1)DNA polymerase contains a 2nd active site that acts as an exonuclease that promotes degradation of single stranded DNA from its 3' OH end into mononucleotides.
2)Contains a 3rd active site that degrades double stranded DNA 5' to 3' (its natural substrate is RNA primers). |
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Term
| Important auxiliary factors/proteins in DNA replication |
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Definition
1)SINGLE STRANDED DNA BINDING PROTEIN
-Stretches out/Straightens out DNA template. Also prevents ssDNA from annealing with each other.
2)THE SLIDING CLAMP
-Tether polymerase to DNA strand. Clamp loader and two halves of sliding camp are formed with addition of ATP.
3)DNA HELICASE
-breaks H-bond of dsDNA. Requires ATP.
4)DNA PRIMASE
-RNA polymerase can initiate strands DE NOVO, places down small strands (~10 RNTPs). It can also use dNTPs but the concentration of rNTPs are higher in the cell.
5)DNA LIGASE
-Joins adjacent 3'-OH and 5'Phosphate groups otherwise the strands will fall off during the next cycle of replication. Likes to ligate DNA-DNA, not RNA-DNA. Requires ATP.
6)TOPOISOMERASE
-Transiently nicks and ligases DNA strand to relieve torsional strain as a result of supercoiling. Since it has ligase activity, it also requires ATP. |
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Term
| Problems with DNA Replications |
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Definition
1)How is a primer added? DNA can only add to the 3' end of existing strands (DNA primase)
2)How will the anti-parallel information strand get synthesized? DNA polymerase cannot add to the 5' of an existing strand. (DNA primase)
3)Separating the parent strands is energetically unfavorable. (DNA helicase)
4)How can the discontinuous strands be linked? (DNA ligase)
5)How can you relieve torsional strain caused from the supercoiling of DNA replication? (Topoisomerase) |
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Term
| Leading and lagging strand |
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Definition
Leading strand is synthesized CONTINUOUSLY
Lagging strand is synthesized DISCONTINUOUSLY (Okazaki fragments) |
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Term
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Definition
1)Recognize and open up DNA
2)Establish RNA primers followed by DNA elongation.
3)Two forks grow in opposite directions. |
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