DNA REPLICATION AND REPAIR

1. DNA REPLICATION AND REPAIR 4.3

2. Cell Division • In eukaryotic cells, genetic material in the nucleus is divided EQUALLY between two daughter nuclei. • THIS IS CALLED MITOSIS (involves nuclei division) • The cell is split into two (along with the new nuclei) • Cytokinesis: the CYTOPLASM is split into two (along with the new nuclei)

4. There are two types of cell division... Mitosis: • occurs in SOMATIC cells. • Somatic cell: every cell in a multicellular organism EXCEPT for the sex cells. • Essential for growth of tissue, growth of a fetus/child, skin cell replacement, repair of damaged tissue, etc. • Meiosis: • Occurs in the sex cells (eggs and sperm) • The egg cell and sperm cell each contribute to ½ the DNA to make offspring.

5. Let’s focus on Mitosis. • Going back to the cell theory... • Every cell originates from another cell. • How does this happen!? • Each daughter cell (new skin cell) has an exact copy of the parent cell’s DNA. • The DNA must be able to replicate. • each of the complimentary DNA strands can act as a TEMPLATE to build another complimentary strand, resulting in two identical DNA molecules.

6. Semiconservative vs. Conservative Replication • What was known: • DNA was the hereditary molecule. • DNA had to replicate somehow. • Question: • Was the parental DNA split between the two daughter cells? • Did one daughter cell contain the DNA from the parent as the other contained copies of that parent?

7. This diagram is in your textbook. Can you point out what is wrong with it?! (Think critically. Don’t believe everything that you see/hear)

8. Meselson & Stahl • Came up with a clever experiment to suggest that DNA replicated in a SEMICONSERVATIVE way • Half of the parental DNA was in each daughter cell. • E. coli was grown in 15N (______ nitrogen) The organism would use the form of N available when it forms new DNA. • Nitrogen is a major component of _____________. • Reproduced by division for 17 generations. • Both DNA strands were labelled with 15N. • Transferred this ________ N – containing strain to a ‘normal’ 14N medium and let the bacteria multiply. • Extracted DNA from E.coli and centrifuged after each generation of growth. • Used DENSITY GRADIENTS of the DNA to determine if conservative or semiconservative.

9. If Conservative... The centrifuged test tubes of each generation would show the following results: First generation: A ‘heavy’ band and a ‘light’ band, both of approximately equal widths. Second generation: A ‘heavy’ band and a ‘light’ band, with the ‘light’ band being 3X the width of the heavy band.

10. If Semiconservative... First generation: Only one intermediate band. Second generation: One light band and one intermediate band of equal width.

11. Meselson and Stahl’s Results: before being switched to normal N. First generation of being normal N. Second generation of being in normal N. THEREFORE, REPLICATION OCCURS ________________.

12. Prokaryotic vs. Eukaryotic DNA • Prokaryote • One circular chromosome • Replication is simpler than in eukaryotes. • One origin of replication. • Eukaryote • Many chromosomes in strand form • Replication is similar to prokaryotes but more complex. • Many origins of replication

13. Two major phases of DNA replication: Separating the DNA strands Building the complimentary strands

14. 1) Strand Separation Goal: to expose a template strand. • DNA helicase • unwinds the double helix by breaking the H bonds between complementary base pairs. • Single-stranded binding proteins (SSBs): • Anneal: pairing of complementary strands of DNA through hydrogen bonding. • The two strands have a natural propensity to anneal. • SSBs bind to DNA strands and block H-bonding. • DNA gyrase • Relieves tension of unwinding DNA • Works by cutting both strands of DNA, strands can swivel around each other, and then resealed.

16. Replication Forks • Replication begins in two direction from the origin(s) as the region of DNA is unwound. • Cannot be fully unwound: DNA is large compared to the cell. It’s just packed really tight when not replicating. • Diameter of cell: 5um. • Length of human chromosome: 1 cm (2000 x) • Replication fork: the region where the enzymes replicating a DNA molecule are bound to untwisted, single-stranded DNA. • Eukaryotes: usually multiple replication forks on DNA molecule  rapid replication • Prokaryotes: usually only one point of origin. • DNA replication proceeds toward the direction of the replication fork in one strand, and away from the replication fork in the other. • Replication bubble: when two replication forks are near each other.

17. Eventually, the replication bubbles fuse and two daughter DNA molecules are formed.

18. 2) Building Complementary Strands • Prokaryotes: • DNA polymerase I, II, and III: enzymes for replication and repair. • DNA polymerase III: responsible for synthesizing complementary strands of DNA during replication. • Eukaryotes: five DNA polymerases.

19. RNA primer & Primase • DNA polymerase cannot initiate a complimentary strand on its own. • Requires a 3’ starting point. • 10-60 temporary base pairs of DNA annealed to the template strand by primase. • NOW DNA polymerase III can start elongation of the complementary strand.

20. DNA polymerase III • Synthesizes DNA in 5’  3’ direction. • Adds free deoxyribonucleosidetriphosphates to the 3’ end. • Deoxyribonucleosidetriphosphates: free bases in the nucleoplasm. • Energy for building the complimentary strand? • Bond between first and second phosphate is broken. • Drives the dehydration synthesis: adding complementary nucleotide to the elongating strand. • Two phosphates recycled.

21. DNA is always synthesized in the 5’  3’ direction • Template strands run antiparallel • Leading strand: the new strand of DNA that is synthesized continuously. • uses the 3’  5’ template strand as guide. • Built towards the replication fork. • Can be built continuously. • Lagging strand: the new strand of DNA that is synthesized in short fragments, which are later joined together. • uses the 5’  3’ template strand as guide. • Built away from the replication fork. • Must be built in short fragments...

22. Okazaki Fragments & The Lagging Strand Short fragments of DNA that are a result of the synthesis of the lagging strand. Primers must be continuously added near the replication fork. DNA polymerase III builds okazaki fragments.

23. DNA polymerase I Removes RNA primers from the leading and lagging strand. Replaces them with appropriate deoxyribonucleotides.

24. DNA ligase Joins Okazaki fragments into one strand by the creation of a phosphodiester bond. THE TWO NEW STRANDS OF DNA ARE SYNTHESIZED AND AUTOMATICALLY TWIST INTO A HELIX.

27. Quality Control • DNA polymerase I and III act as quality control checkers • ‘proofread’ newly synthesized strand. • Woops! There’s a mistake! • Either enzyme can act as an EXONUCLEASE. • Backtracks, excises the incorrect nucleotide, and replaces it. • If error missed by exonuclease, may be corrected by one of the several repair mechanisms that operate after the completion of DNA replication.

28. http://www.youtube.com/watch?v=teV62zrm2P0&feature=related

29. Seatwork/Homework Look at the summary on page 222. Make sure you understand every point, and the diagram shown. Page 223, #1-8.