1. DNA Replication

2. Warm-UP  What is the cell cycle? When does DNA replicate during the cell cyle?

3. Learning Goals  Determine the mechanism of DNA replication as semiconservative and involving many different enzymes and proteins.  Identify proofreading/error correcting ability of DNA polymerases.  Determine how errors in replication can lead to genetic changes. GET OUT YOUR DNA MODELS!

4. The Cell Cycle  it is a fundamental property of cells to be able to reproduce. This is accomplished through the cell cycle, which is the order of events that occur for a cell to divide to produce two daughter cells.  Scientists knew that there had to be a way for the genetic material to be copied and transmitted to each daughter cell. We now know this occurs during the S phase of the cell cycle, which occurs before methods of cell and cytoplasmic division like mitosis/meiosis.

5. DNA Replication  After elucidating the structure of DNA, a model for replication was needed. Scientists had their thoughts, but evidence was needed. Three potential models were proposed:  Conservative model: one completely new molecule, one conserved parent molecule  Semi-conservative model: Both molecules are made up of a parent strand and a new strand  Dispersive model: Hybrid strands, each being a mixture of parent and daughter

6. A Clever Experiment  Matthew Meselson and Franklin Stahl reasoned that they could test the models if they could distinguish between parent and daughter strands of DNA. They used two forms of nitrogen (the lighter 14 N and the heavier 15 N to distinguish).  https://www.youtube.com/watch?v=WsW2BZiLdpY https://www.youtube.com/watch?v=WsW2BZiLdpY

7. Semi-Conservative Requirements  As it was determined through the experiment that DNA is replicated semi- conservatively, a method of separating the two parent strands is needed.  This begins at a site called the origin of replication (ori), and involves the use of special enzymes which pull the strands apart creating a replication bubble.  Once strands are separated, new daughter strands can begin to grow. This occurs in both directions from the origin of replication at special sites called replication forks.

8. Unzipping  DNA Helicase is an enzyme that is able to hydrolyse ATP in order to break hydrogen bonds between complementary bases.  Bases would normally anneal (hydrogen bonds would reform), but special proteins called single stranded binding proteins (SSBs) stop this from happening A third type of enzyme, topoisomerase, can regulate the amount of tension in the DNA double helix, as breaking the hydrogen bonds can overwind the DNA ahead of the replication fork.

9. Elongating  Once the strands are separated at the origin of replication, an enzyme called DNA Polymerase III can begin adding dNTPs to their complementary bases on the parent strand.  There are problems that arise however:  DNA Polymerase can only catalyze the addition of a dNTP to the free 3’OH of a preceding base. Use your models to visualize the issues that Polymerase III has.

10. Polymerase Needs Help  To overcome the restrictions that DNA Polymerase III has, many enzymes are used: Primase: An enzyme that lays down a short RNA sequence that provides a free 3’OH for Polymerase III to start. DNA Polymerase III: Begins replicating in a 5’ to 3’ direction. DNA Polymerase I: Replaces RNA Primer with DNA sequence DNA Ligase: Seals up any breaks in the sugar phosphate backbone of the new strand.

11. Leading Strand and Lagging Strand  One strand of the DNA is built continuously (as the direction of polymerization is the same as the growing fork). This is called the leading strand.  The other strand needs to be built discontinuously, in short, choppy segments. This is called the lagging strand. The short segments that arise on the lagging strand are called Okazaki fragments. When looking at the two forks that result from one replication bubble, what is the leading strand at one fork is the lagging strand in the other (as the forks are moving in opposite directions, but polymerase has to build only from 5’ to 3 ’

12. Termination  Termination occurs once the synthesis of the new DNA strands is complete. Two new DNA molecules separate from each other and the replication machinery is dismantled. The chromatin molecule can have thousands of origins of replication, which all grow into each other, allowing for faster replication.

13. Errors in Replication  DNA polymerase enzymes are amazingly particular with respect to their choice of nucleotides during DNA replication. Nonetheless, these enzymes do make mistakes at a rate of about 1 every 100 000 nucleotides. In a human, with ~ 6 billion base pairs in each diploid cell, that would amount to about 120 000 mistakes every time a cell divides.  Some mistakes are corrected immediately during replication through a process known as proofreading, and some are corrected after replication in a process called mismatch repair.

14. Proofreading  When an incorrect nucleotide is added to the growing strand, replication is stalled by the fact that the nucleotide’s exposed 3’-OH group is in the “wrong” position. Polymerase III can recognize this and replace the incorrect nucleotide with the correct one. This fixes about 99% of errors.

15. Mismatch Repair  After replication, incorrectly paired nucleotides cause deformities in the secondary structure of the final DNA molecule. During mismatch repair, enzymes recognize and fix these deformities by removing a section containing the incorrectly paired nucleotide and replacing it with the correct one.

16. Mutations  Errors that remain after DNA polymerase proofreading and mismatch repair are considered mutations once cell division occurs.  If this occurs in cells that give rise to gametes, they can be transmitted to offspring. http://youtu.be/FBmO_rmXxIw?t=4m57s

17. Review  Create a summary table with the following enzymes/proteins and their functions: DNA Helicase, single strand binding proteins, DNA Polymerase I, DNA Polymerase III, DNA Ligase, Primase  Compare the mechanisms by which the leading and lagging strands are replicated. Explain why it is necessary for two mechanisms to exist.  Why do you think it is necessary for eukaryotic DNA to have multiple replication origin sites?  What do you think might happen if the DNA that provided instructions for making mismatch repair enzymes was mutated?  What do you think might happen if the DNA that provided instructions for cell cycle regulating proteins were mutated?