Agenda 2/23 DNA Replication Notes DNA Replication illustrations Homework 1. DNA Replication Video and Notes 2. Chp 12 Reading and Notes (due Monday) Turn in: Video Notes, Chp 16 if finished

When would DNA need to replicate? Brainstorm 4 specific times when DNA replication would be needed. https://youtu.be/TNKWgcFPHqw

Possible Ways DNA Replication Occurs

Experiment to show semi-conservation replication (Meselton and Stahl)

How does DNA replication occur? 1. Strands must separate (hydrogen bonds broken) 2. Each old strand serves as a template for the new strand (new base pairing) Result: 2 molecules of DNA each with 1 ‘old’ strand and 1 ‘new’ strand

Prokaryotic DNA Replication The prokaryotic chromosome attaches to the plasma membrane. The DNA is then replicated in both directions. Emphasize that the chromosome of a prokaryotic cell is circular as opposed to linear as in eukaryotic cells. Only one origin of replication which attaches to the plasma membrane is present in prokaryotic cells. Replication of the prokaryotic chromosome occurs in both directions as does replication of eukaryotic chromosomes. Emphasize that prokaryotes have far fewer DNA base pairs than eukaryotes. E. coli has about 4.6 million base pairs whereas a human eukaryotic cell has 3 billion base pairs to replicate.

Eukaryotic DNA Replication DNA replication occurs simultaneously in many locations along the very long eukaryotic chromosomes. As stated previously, DNA replication occurs in both directions within eukaryotic cells. The site where it begins is called the origin of replication. Depending on the source, it has been reported that DNA elongation occurs at a rate of 350-1000 nucleotides added per second in eukaryotic cells. Note in E. coli there are 4.6 million base pairs in its single, circular chromosome and only one origin of replication. In human somatic cells, there are three billion base pairs, which is FAR more than present in prokaryotic cells. It would take too long to have only one origin of replication in replicating the DNA. It is estimated that there are 5 × 104 replication origins among a human’s 23 chromosomes containing 3 × 109 base pairs. Three replication bubbles are visible along the DNA within this cultured Chinese hamster cell. The arrows indicate the directions of DNA replication at the two ends of the bubble.

Enzymes of DNA Replication Helicase- breaks the hydrogen bonds between the base pairs Topoisomerase I and II- relieve stress on unwinding by clipping DNA strand ahead of helicase DNA Polymerase III- makes new base pairs with new nucleotides http://www.youtube.com/watch?v=3QWA-tFdGN8

Enzymes of DNA Replication Primase- puts down RNA nucleotides as a ‘primer’ to show DNA Pol III where to start DNA Pol I- replaces RNA primer with DNA nucleotides to finish up replication Ligase- Glues Okazaki fragments together

Direction of DNA Replication DNA Polymerase III can only add nucleotides to a 3’ end of the deoxyribose sugar Because of this, DNA replication can only occur from the 3’ end of the template strand to the 5’ end of the template strand Synthesis of new nucleotides occurs in the 5’ to 3’ direction Problem: Anti-parallel strands!

Okazaki Fragments On the 3’5’ template strand, DNA pol III can synthesis new DNA continuously (leading strand) But, the 5’3’ template stand has to wait for helicase to open up more DNA and DNA Pol III works in reverse (lagging strand) This results in ‘fragments’ of new DNA synthesized on the lagging strand (Okazaki Fragments) DNA Ligase is used to glue fragments together to complete the new strands

The Lagging Strand and Ligase Students are responsible for knowing ligase and its role in DNA replication. Approach this example with caution, but if student’s know the name of the procedure when a female human has her “tubes tied” formally named a “tubal ligation”, it helps them remember both the name and function of this ligase enzyme as it relates to DNA synthesis. This animation, shows the leading strand being synthesized followed by the lagging strand. The enzyme named ligase ties them together.

Simplified Steps of DNA Replication 1. Helicase separates strands of DNA while topoisomerase ‘cuts’ strand so helicase can separate strands more easily 2. Primase lays down RNA nucleotide primers to tell DNA Pol III where to start 3. DNA Pol III will lay down new DNA nucleotides in 5’-> 3’ direction This creates a leading strand and lagging strand 4. Lagging strand Okazaki fragments will be glued together by ligase 5. DNA Pol I replaces RNA primers with DNA nucleotides

DNA Replication Animation https://youtu.be/TNKWgcFPHqw

DNA Replication Drawing Draw a molecule of DNA replicating and label: Ligase DNA polymerase Primase Helicase Topoisomerase Leading Strand Lagging Strand Okazaki Fragments New Strand/Old Strand 5’ and 3’ ends of all strands Draw the enzymes as something that relates to its function (paint brush, glue bottle etc.) Show RNA Primers in different color than DNA nucleotides Show new strand and old strand in different colors Write steps to replication on your picture in your own words!