Covalently bonded sugar-phosphate backbone with relatively strong bonds keeps the nucleotides in the backbone connected in the correct sequence.

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1 Unit 14: DNA Replication Study Guide U7.1.1: DNA structure suggested a mechanism for DNA replication (Oxford Biology Course Companion page 347). 1. Outline the features of DNA structure that suggested a mechanism for DNA replication. Complimentary base pairing ensures that the genetic message is conserved when each strand replicates. Covalently bonded sugar-phosphate backbone with relatively strong bonds keeps the nucleotides in the backbone connected in the correct sequence. Hydrogen bonds between strands allows strands to separate relatively easily for replication. U2.7.1: The replication of DNA is semiconservative and depends on complementary base pairing (Oxford Biology Course Companion page 111). 2. Describe the meaning of semiconservative in relation to DNA replication. Products of DNA replication each contain one of the original strands and one new strand of DNA. 3. Explain the role of complementary base pairing in DNA replication. Complimentary base pairing ensures the sequence remains consistent after DNA replication. You only need one strand to create exact replica of the two stranded original.

2 S2.7.2: Analysis of Meselson and Stahl s results to obtain support for the theory of semi-conservative replication of DNA (Oxford Biology Course Companion page 113). 4. Compare dispersive, conservative and semi-conservative replication. Semi-conservative: each copy has one original and one new strand Conservative: original DNA and new DNA Dispersive: Original DNA broken into segments and interspersed with new DNA 5. Predict experimental results in the Meselson and Stahl experiment if DNA replication was dispersive, conservative or semi-conservative. Semi-conservative: There would be one band of 15 N after cycle 1 and two bands ( 14 N, 15 N) after cycle 2 (this was observed experimentally) Conservative: There would be two bands after cycle 1. Dispersive: There would be one band after cycle 1 and 2. NOS2.7: Obtaining of evidence for scientific theories- Meselson and Stahl obtained evidence for the semi-conservative replication of DNA (Oxford Biology Course Companion page 112). 6. Describe the procedure of the Meselson and Stahl experiment.

3 E. coli were grown in an isotope of heavy nitrogen ( 15 N) for use in nitrogenous bases. Presence was tested using centrifuge (would settle to the bottom while regular 14 N would be higher in the tube). Transferred cells to 14 N and allowed cells to divide one time and showed one band. Allowed to divide a second time and showed two bands. Start Cycle 1 Cycle 2 Cycle 3 7. Explain how the Meselson and Stahl experiment demonstrated semi-conservative DNA replication It showed the movement of 15N following the pattern of semi-conservative replication (see question 5 & 6) U2.7.2: Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds (Oxford Biology Course Companion page 114). 8. State why DNA strands must be separated prior to replication. Must separate so that each can serve as a template 9. Outline two functions of helicase. Proteins that attach to the DNA double helix and separate the strands by breaking the hydrogen bonds. Also separates self-annealed RNA molecule from DNA. 10. State the role of the origin of replication in DNA replication. The sequence at which replication is initiated

4 11. Contrast the number of origins in prokaryotic cells to the number in eukaryotic cells. Prokaryotic: one origin and two forks Eukaryotic: multiple origins with many bubbles of replication that will eventually fuse. U7.1.4: DNA replication is carried out by a complex system of enzymes (Oxford Biology Course Companion page 349). 12. Outline the role of the following proteins in DNA replications: helicase, topoisomerase (AKA gyrase), single stranded binding proteins, primase, DNA polymerase III, DNA polymerase I, and DNA ligase. Helicase: opens the DNA helix by breaking hydrogen bonds Topoisomerase (gyrase): helps relieve tension stress on DNA when unwinding Single Stranded Binding Proteins (SSBPs): prevent parent DNA strands from reconnecting Primase: synthesizes RNA primers to start replication DNA Polymerase III: main enzyme that builds complimentary strand by adding nucleotides in a 5 3 direction. DNA Polymerase I: removes the RNA primer and replaces it with DNA nucleotides Ligase: covalently bonds the sugar-phosphate backbone between adjacent Okazaki fragments on the lagging strand.

5 U2.7.3: DNA polymerase links nucleotides together to form a new strand, using a pre-existing strand as a template (Oxford Biology Course Companion page 115). 13. Describe the movement of DNA polymerase along the DNA template strand. Daughter strand is built from 5 3, parent strand/template is read from Describe the action of DNA polymerase III in pairing nucleotides during DNA replication. It reads the parent strand and adds complimentary nucleotides to build a new strand of DNA. Can only add nucleotides to the 3 end. Adds the 5 end of incoming nucleotide to the 3 end of the previously placed strand. U7.1.5: DNA polymerases can only add nucleotides to the 3

6 end of a primer (Oxford Biology Course Companion page 350). 15. Explain the need for RNA primers in DNA replication. Short strand of RNA (10 bases) that acts as a starting point for DNA replication. Needed because DNA polymerase III can only add nucleotides to existing strand. 16. Explain what is meant by DNA replication occurring in a 5' to 3' direction. The new DNA strand is built from its 5 end towards its 3 end. U7.1.3: DNA replication is continuous on the leading strand and discontinuous on the lagging strand (Oxford Biology Course Companion page 349). 17. Compare replication on the leading strand and the lagging strand of DNA. Leading strand: DNA polymerase III moves in same direction as helicase and adds complimentary nucleotides to the daughter strand. Lagging strand: DNA polymerase III moves in opposite direction as helicase 18. Explain why replication is different on the leading and lagging strands of DNA. Because DNA polymerase III can only add new nucleotides to the 3 of the growing strand, replication runs in opposite directions on the leading and lagging strands. 19. Outline the formation of Okazaki fragments on the lagging strand. Because DNA polymerase III is moving away from the

7 replication fork on the lagging strand, it must build DNA in short sections called Okazaki fragments. U3.5.2: PCR can be used to amplify small amounts of DNA (Oxford Biology Course Companion page 188). 20. State the function of the PCR. Polymerase Chain Reaction is used to make many copies of a specific region of DNA, making millions of copies of a particular DNA sequence. 21. Describe the selectivity of the PCR. A specific section of DNA can be copied using PCR. By using primers that are specific to that section, only the wanted region will be copied. A2.7.1: Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR) (Oxford Biology Course Companion page 115). 22. Outline the process of the PCR. 1. Heat is used to denature the DNA 2. Temperature is lowered to allow the primer to anneal (bind) to the DNA. 3. Taq Polymerase attaches to the primer and binds complimentary strand of DNA

8 4. Repeat to make many copies 23. Explain the use of Taq DNA polymerase in the PCR. Taq polymerase is a type of DNA polymerase named after the bacteria Thermus aquaticus in which it was discovered. The bacteria lives in hot springs so it can withstand high temperatures required in PCR. Other DNA polymerase molecules would denature in the heat.

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