“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 onstate.edu/people/christ opher-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 12-1 T 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?

Page 89 Figure 5-13 Demonstration of the semiconservative nature of DNA replication in E. coli by density gradient ultracentrifugation.

Table 30-1Properties of E. coli DNA Polymerases. Page 1145

DNA Polymerases Enzymes 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) terminal transferases). and even enzymes that make DNA without using a template (terminal 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 5-31Action of DNA polymerases. Page 99 DNA polymerases assemble incoming deoxynucleoside triphosphates on single-stranded DNA templates such that the growing strand is elongated in its 5’  3’ direction.

Figure 5-32aReplication of duplex DNA in E. coli. Page 100

Figure 5-32bReplication of duplex DNA in E. coli. Page 100 Animation

Figure 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 NTP B stabilizes the negative charges on NTP

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

Figure 30-7Priming of DNA synthesis by short RNA segments. Page 1139

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 activity 2. 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 30-8bX-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). Page 1141

Figure 30-12Nick translation as catalyzed by Pol I. Page 1144

Figure 30-8a X-Ray structure of E. coli DNA polymerase I Klenow fragment (KF) in complex with a dsDNA. Page 1141

Here’s a computer model Here’s a computer model Overview of DNA and replication Another oneAnother one with review questions t/animations/dna_replication/index.html This is a pretty good outline:

Figure 30-13a X-Ray structure of the  subunit of E. coli Pol III holo- enzyme. Ribbon drawing. Page 1146

Figure 30-13bThe  subunit of E. coli Pol III holoenzyme. Space-filling model of sliding clamp in hypothetical complex with B-DNA. Page 1146

ev/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 30-14Unwinding of DNA by the combined action of DnaB and SSB proteins.

Figure 30-15Electron microscopy–based image reconstruction of T7 gene 4 helicase/primase. Page 1147

Figure 30-17Active, 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. Page 1149

Figure The reactions catalyzed by E. coli DNA ligase. Page 1150

Figure X-Ray structure of DNA ligase from Thermus filiformis. Page 1151

Figure 30-22X-Ray structure of E. coli primase. Page 1151