BCOR 101

Sept 9, 2004

Replication overview

•General problem is how to maintain integrity of the DNA sequence? (think of the game “telephone”)

•Need to:

–unwind DNA, add an RNA primer, find an appropriate base, add it to the growing DNA fragment, proofread, ligate fragments together, remove the initial primer, fill in the gap with DNA

•All of this is fast, about 100 bp/second

• animation from DNAi

•Meselson-Stahl experiment

–Grow bacteria with 15N, then transfer to regular media (14N) for several generations.

–Extract DNA and measure its density each generation.

–What do you predict?

•Gen 0 ?

•Gen 1 ?

•Gen 2 ?

Compare your predictions to figure in the book. Convince yourself that semi-conservative replication is the only model that will work.

Harlequin chromosomes provide visual evidence of semi-conservative replication

•It is possible to label DNA by using the base analog 5BdU, which can substitute for T but stains only lightly.

•Look at the picture in your book and convince yourself that semi-conservative replication is the only model consistent with the results.

–What results would they see after only 1 round of synthesis with 5BdU? After 3 rounds?

–Interestingly, a similar expt was published by Taylor? Et al in 1957, a year before the Meselson-Stahl expt. However, the Meselson-Stahl expt is the one textbooks all cite. Which type of expt do you find most convincing?

Bacterial replication

•Circular DNA

•Single origin or replication

•Bi-directional

Fig. 3.10 Bidirectional replication of circular DNA molecules

Fig. 3.11b Diagram showing the unreplicated, supercoiled parent strands and the
portions already replicated

So, how is the chromosome replicated?

Fig. 3.4 DNA chain elongation catalyzed by DNA polymerase

Understand how a nucleotide is added to the chain and be able to draw the reaction (at least in stick figure form)

First look at leading strand:

•Helicase unwinds a bit of DNA, single-strand binding proteins keep the strands apart.

•Primase adds an RNA primer

•Pol3 adds complementary nucleotides to 3’ OH and slides down the molecule, continuing to extend the sugar-phosphate chain.

•This continues until it gets all the way around the molecule, or until it reaches a termination sequence.

•Gyrase relieves tension as needed.

•(E. coli has about 40,000 turns in the circular chromosome-- each of them must be unwound to separate the DNA strands)

Now look at lagging strand

•Can’t form continuous molecule, since synthesis only happens 5’ to 3’. SO, need many primers and lots of short fragments

•Primase adds an RNA primer and Pol3 adds nucleotides until it reaches the previous primer

•Pol1 removes RNA primer on adjacent stand and adds dNTPs

–Q: why can’t Pol3 remove the RNA?

•Ligase connects the fragments

–Q: why can’t Pol1 make the final connection?

To do:

•Draw a replication bubble for E. coli, label the bases on the template, primer, and newly synthesized dna using the shorthand DNA notation.

•Convince yourself that synthesis must go 5’ to 3’ and that replication must be semi-discontinuous.

•Study and understand the animations about replication on your textbook CD

•Look again at the animation from DNAi

–http://www.dnai.org

–(go to the section on copying the code)