Replication of DNA Notes & Animations

Cell Cycle G1: Growth S: Synthesis of DNA (Replication) G2: Growth, preparation of organelle for mitosis M: Mitosis

How does DNA Replicate? Meselson and Stahl’s experiment: Hypothesis 1: “conservative” replication Hypothesis 2: “semi-conservative” replication Hypothesis 3: “dispersive” replication

“Conservative” Replication

“Semi-Conservative” Replication

“Dispersive” Replication

Method E. coli grown in 15N (heavy Nitrogen) medium. Allowed to reproduce for 17 generations. (Makes all DNA heavy.) Then, transferred to 14N media & allowed to reproduce again (New DNA will be light) Centrifuged (spun) so that “heavy” DNA sinks to bottom, “light” DNA stays on top.

Predictions “Conservative” Replication: After 1 replication, 2 distinct bands original “heavy” DNA at bottom, new “light” DNA on top After 2 replications, same 2 bands, but more DNA in “light” band

Conservative Replication Heavy DNA Light DNA

Predictions “Semi-Conservative” Replication After 1 generation, 1 band in middle. After 2 generations, 1 light band, 1 middle band

Semi-Conservative Replication Heavy DNA Light DNA

Predictions “Dispersive” replication: After 1 replication, band would be in the middle. After 2 or more replications, band would be in the middle, but getting higher each time. Always hybrid.

Dispersive Replication Prediction Heavy DNA Light DNA

Observations

Now we know… Replication consists of 3 steps: Initiation: DNA helicase unwinds double helix by breaking H-bonds Proteins hold DNA apart (if not, DNA would re-anneal – stick back together) Replication begins in 2 directions from many origins in eukaryotes – speeds up process

Replication Bubbles

Replication: Step 2 Elongation: Requires 4 more enzymes (eukaryotes) Primase Makes RNA primer DNA polymerase III Can’t start from nothing – attaches to primer Moves in a 5’ to 3’ direction continuously (adds nucleotides to 3’C)

Uh oh, what about 3’  5’? Made in short fragments called Okazaki fragments Consist of RNA primer, then DNA polymerase adds a short sequence, then start over Called the “lagging” strand 5’  3’ called the “leading” strand

Elongation, continued… DNA polymerase I Replaces RNA primers with DNA nucleotides and proofreads the new strand DNA ligase Joins Okazaki fragments together

Proofreading When mistake is found, it is cut out and replaced with correct base (Pol I) Errors missed at a rate of 1/billion bases!

Step 3 of Replication Termination Helix re-anneals automatically. Note: strands can’t be fully finished because DNA polymerase I cannot replace the primers at the ends of the DNA. Ends of chromosomes are stretches of repetitive “junk” DNA called Telomeres Telomeres extended by telomerase (expressed during meiosis, and by cancerous cells) Chromosomes lose about 100 bases from this telomeric region every replication