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“A role for aneuploidy in genome evolution?”
Andreas Madlung, Associate Professor,
Biology Dept., University of Puget Sound, Tacoma, WA
Wednesday, April 8, 2009 at 4 pm in
BI 234
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Prof. Chris Mathews
DNA Precursor Metabolism and Genomic Stability
Oregon State Univ.
Department of Biochemistry and Biophysics http://biochem.science.ore
gonstate.edu/people/christopher-k-mathews
Chemistry Seminar F 4/10 3:15 pm SL 130
Do try to attend. This guy is good!
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Office Hours this week:
M 12-1T 3–4 pm
R 2-3
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How would YOU go about determining the mechanism
of DNA replication?????
What would a geneticist do?
What would a biochemist do?
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Figure 5-13 Demonstration of the semiconservative nature of DNA replication in E. coli by density gradient ultracentrifugation.
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Table 30-1 Properties of E. coli DNA Polymerases.
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DNA PolymerasesEnzymes that replicate DNA using a DNA template are called DNA polymerases.
However, there are also enzymes that synthesize DNA using an RNA template (reverse transcriptases) and even enzymes that make DNA without using a template (terminal transferasesterminal transferases).).
Most organisms have more than one type of DNA polymerase (for example, E. coli has five DNA polymerases), but all work by the same basic rules.
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1. Polymerization occurs only 5' to 3'2. Polymerization requires a template to copy: the complementary strand.3. Polymerization requires 4 dNTPs: dATP, dGTP, dCTP, dTTP (TTP is sometimes not designated with a 'd' since there is no ribose containing equivalent)4. Polymerization requires a pre-existing primer from which to extend. The primer is RNA in most organisms, but it can
be DNA in some organisms; very rarely the primer is a protein in the case of certain viruses only.
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Figure 5-31 Action of DNA polymerases.
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DNA polymerases assemble incoming deoxynucleoside triphosphates on single-stranded DNA templates such that the growing strand is elongated in its 5’ 3’ direction.
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Figure 5-32a Replication of duplex DNA in E. coli.
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Figure 5-32b Replication of duplex DNA in E. coli.
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Animation
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Figure 30-10 Schematic diagram for
the nucleotidyl transferase
mechanism of DNA
polymerases.
A and B are usually Mg+2 divalent metal
A activates the primer 3’OH for nucleophillic attack on -phosphate of NTPB stabilizes the negative charges on NTP
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Figure 30-28The replication of E. coli DNA.
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Figure 30-7 Priming of DNA synthesis by short RNA
segments.
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DNA Polymerase I (pol I) from E. coli first DNA polymerase characterized. approximately 400 molecules of the enzyme per cell.
large protein with a molecular weight of approximately 103 kDa (103,000 grams per mole). a divalent cation (Mg++) for activity
Three enzymatic activities:1. 5'-to-3' DNA Polymerase activity2. 3'-to-5' exonuclease (Proofreading activity)3. 5'-to-3' exonuclease (Nick translation activity)
It is possible to remove the 5'-to-3' exonuclease activity using a protease to cut DNA pol I into two protein fragments
Both the polymerization and 3'-to-5' exonuclease activities are on the large Klenow fragment of DNA pol I, and the 5'-to-3' exonuclease activity is on the small fragment.
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Like all known DNA polymerases, DNA polymerase I requires a primer from which to initiate replication and polymerizes deoxyribonucleotides into DNA in the 5' to 3' direction using the complementary strand as a template.
Newly synthesized DNA is covalently attached to the primer, but only hydrogen-bonded to the template.
The template provides the specificity according to Watson-Crick base pairing 4.
Only the alpha phosphate of the dNTP is incorporated into newly synthesized DNA
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Figure 30-8b X-Ray structure of E. coli DNA polymerase I Klenow fragment (KF) in complex with a dsDNA (a tube-and-arrow representation of the complex in the same orientation as Part a).
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Figure 30-12 Nick translation as catalyzed by Pol I.
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Figure 30-8aX-Ray structure of E. coli DNA polymerase I
Klenow fragment (KF) in complex with a
dsDNA.
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Here’s a computer modelhttp://www.youtube.com/watch?v=4jtmOZaIvS0
Overview of DNA and replication
http://207.207.4.198/pub/flash/24/menu.swf
Another one with review questions
http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html
This is a pretty good outline:http://www.youtube.com/watch?v=teV62zrm2P0&NR=1
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Figure 30-13a
X-Ray structure of the subunit of E. coli
Pol III holo-
enzyme. Ribbon
drawing.
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Figure 30-13b The subunit of E. coli Pol III holoenzyme. Space-filling model of sliding clamp in hypothetical complex with B-
DNA.
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http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/poliiib_2/poliiib.htm
Sliding clamp
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Clamp loading:·All clamp loaders utilize the energy of ATP to assemble their respective clamps onto replication forks·Various studies have suggested that the clamp loading complex starts off in a closed form and, upon bind ATP, is drven into an open conformation that binds the clamp ( dimer)One formed, this complex between the clamp loader and the clamp binds to the DNA, inserts the DNA through the open clamp and then hydrolyzes ATP
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Figure 30-14 Unwinding of DNA by the combined action of
DnaB and SSB proteins.
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Figure 30-15Electron microscopy–based image reconstruction of T7 gene 4 helicase/primase.
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Figure 30-17 Active, rolling mechanism for DNA unwinding
by Rep helicase.
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Figure 30-19X-Ray structure of the N-
terminal 135 residues of E. coli SSB in complex with
dC(pC)34.
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Figure 30-20The reactions
catalyzed by E. coli DNA ligase.
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Figure 30-21
X-Ray structure of DNA ligase from
Thermus filiformis.
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Figure 30-22 X-Ray structure of E. coli primase.P
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