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Warm-up

Warm-up. Which parts of the nucleotides are connected to make one strand (half) of DNA? Which parts of the nucleotides connect the two strands (halves) of DNA? What type of bond connects the two strands together? If a DNA molecule is 30%Adenine how much Cytosine is there?.

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Warm-up

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  1. Warm-up • Which parts of the nucleotides are connected to make one strand (half) of DNA? • Which parts of the nucleotides connect the two strands (halves) of DNA? • What type of bond connects the two strands together? • If a DNA molecule is 30%Adenine how much Cytosine is there?

  2. What does DNA look like? • We can describe its molecular structure • Next week we will see what DNA looks like after isolation.

  3. DNA replication When a cell divides where Does the extra DNA come from?

  4. DNA Replication Whenever a cell divides into two new cells, it needs to make an exact copy of the DNA. What would happen if a cell divided without DNA making a copy of itself? The process of copying DNA is calledDNA replication.

  5. DNA Replication 1. • Replication could be: • Conservative: Old strands stay together and act as a template. • Semi conservative: Strands split and act as a template. • Dispersive: Strands separate AND break apart and act as templates.

  6. Possible DNA Replication Models

  7. The Meselson-Stahl Experiment • E. coli bacteria were cultured for several generations in heavy nitrogen (15N - normal nitrogen is 14N) (Why is 15N heavier than 14N?). • The bacteria incorporated 15N into their nucleotides and thus, their DNA • Meselson and Stahl then transferred the bacteria to a medium containing 14N, • So…. any DNA that the bacteria synthesized would be lighter than the "old" DNA made with the heavier 15N medium • The DNA was extracted from the cells and centrifuged in a cesium chloride density gradient for 20 hours at 40,000rpm. • The DNA migrated to a point that was equivalent to their density • http://www.dnaftb.org/dnaftb/20/concept/index.html

  8. Possible outcomes

  9. Experimental Results ･Results from the parental generation contained only a single high density band - all DNA molecules contained the "heavy" nitrogen. ･DNA taken from the two generations after the switch contained an intermediate-density band - DNA contained a "heavy" DNA strand from the parent and a complementary "light" DNA strand. ･Density results from generation 3, displayed two bands. They included an intermediate density band, composed of one parental "heavy" strand and a new light band, composed of only DNA strands with "light" nitrogen.

  10. Actual Outcome

  11. So Replication is Semi Conservative

  12. DNA Replication 2 • DNA could be extended in a 3’ -> 5’ direction. • DNA could be extended in a 5’ -> 3’ direction. • DNA could be extended in a both 3’ -> 5’ and 5’ -> 3’ directions simultaneously.

  13. DNA polymerase C A T G C T C A G T T 5’ G T A C G G T C A A DNA synthesis uses the energy released from the triphosphate bond to “power” DNA synthesis… Nucleotides add as 5’ NTPs… Specificity determined by base pairing… “Primer” strand 5’ Template strand Incorrect nucleotide cannot base pair… … and is removed by 3’→5’ exonuclease activity of DNA polymerase… Correct base inserted and 5’→3’ replication continues… Each nucleotide brings the energy needed for its own addition…

  14. 5’ 3’ T T G C C A G T C 5’ G T A C G G T C A A No high energy phosphates 5’-3’ exonuclease 5’ triphosphate If synthesis needs triphosphate bonds can 3’→5’ synthesis be self-correcting? NO! Consider a hypothetical scheme in which DNA synthesis occurs 3’ to 5’… Triphosphates for addition of next nucleotide at 5’ end… “Primer” strand Template strand Incorrect nucleotide cannot base pair… Removed by 5’→3’ exonuclease activity… No high energy phosphates left for insertion of next nucleotide… Replication unable to proceed… 3’→5’ polymerase cannot be self correcting…

  15. Error: Some comparisons

  16. Therefore:DNA Replication proceeds in a 5’->3’ direction only.

  17. RNA primer of previous Okazaki fragment RNA primer of next Okazaki fragment Fork migration 3’ DNA polymerase can only add to 3’ end of base-paired nucleotide… SO..short lengths of RNA act as “primers” for DNA synthesis… 5’ “Primase” uses DNA template to synthesize a short ( ~10 b) stretch of RNA… Leading strand is only primed at initiation… Each Okazaki fragment of lagging strand begins with new RNA primer… 3’ 5’ 5’ 5’ 3’ 5’ DNA polymerase then elongates the RNA primer by adding dNTPs to its 3’ end… RNA primer removed by “repair exonuclease”… Gap filled by “repair polymerase”… “Ligase” seals nicks to complete strand… 5’ 3’

  18. DNA Replication 3. • Does DNA Replication occur from one point only? OR • Does DNA Replication occur from from many origins?

  19. Replication in Action • 1 human chromosome is an average of 80 million base pairs long (3 billion/46). • If a cell replicates in 10 hours how many bases per second does replication proceed at? • About 2200bp/sec! • E. coli chromosome is 5 million bp, if it replicates every 20 mins how many bases per second does replication proceed at? • About 4200bp/sec

  20. The Speed of DNA Replication • Radioactive nucleotide incorporation has been measured at about 1000bp/sec in prokaryotes and about 50bp/sec in eukaryotes • So DNA replication must occur simultaneously at multiple places along a DNA strand.

  21. “Replication origin” Single strand DNA Origin Replication forks Replication forks Replication forks Replication forks Origins Origins Replication origins: DNA replication begins at specific sites… Initiator proteins (incl helicases) “melt” double helix at origin… ss DNA serves as template for DNA polymerase… 5’ 3’ “Replication bubble” 3’ 5’ “Replication forks” then move outward… Prokaryotes generally use just one origin… Yellow = new DNA Eukaryotes use multiple origins…

  22. Fork migration Fork migration Leading strand template for L fork… Lagging strand template for R fork… Most recently synthesized DNA Lagging strand template for L fork… Leading strand template for R fork… Taking a closer look at replication forks… 5’ 3’ Left fork Right fork 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ The leading strand template for the right fork is the lagging strand template for the left fork... …and vice versa.

  23. DNA Replication 4. • At the replication fork there are MANY enzymes that help in the synthesis of new DNA

  24. Enzymes in DNA replication • Enzymes are proteins that act as machines in the cell to do work. • Many enzymes come together to form a multi-functional replication machine called a Replisome at the replication fork.

  25. DNA replication: a caste of characters… Replication: Helicase: ATP-dependent separation (“melting”) of DNA strands… Single strand binding protein (SSB): binds and stabilizes ss DNA… Primase: synthesizes RNA primers (may be a component of DNA polymerase)… DNA polymerase: synthesizes and “proof-reads” new DNA strand… Repair Exonuclease: removes RNA primer… Repair polymerase: fills “gap” after primer is removed… Ligase: closes “nicks” in DNA Topoisomerase I: creates ss nicks ahead of replication to allow unwinding… Topoisomerase II: creates ds nicks to pass one ds DNA through another… Proof-reading and repair: DNA polymerase: 3’→5’ exonuclease/5’→3’ polymerase… DNA mismatch repair enzymes: exonuclease, polymerase, ligase… DNA Polymerases : 5 known in prokaryotes At least 20 known in eukaryotes.

  26. Complicated picture of replication…

  27. The Whole Enchilada! http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf OR http://www.dnai.org/a/index.html Go to: 1. Copying the code 2. Putting it together 3. Replication

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