“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 

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!

Office Hours this week:

M 12-1T 3–4 pm

R 2-3

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.

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.

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.

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.

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

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

Figure 30-28The replication of E. coli DNA.

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.

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

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

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

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

Figure 30-14 Unwinding of DNA by the combined action of

DnaB and SSB proteins.

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.

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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