1. 11 Chapter 16 The Molecular Basis of Inheritance “We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). This structure has novel features which are of considerable biological interest.” --James Watson and Francis Crick, 1953

2. Watson and Crick After DNA was determined to be the hereditary material, the next question was, how DNA was replicated? The strands are said to be complimentary to each other. That is, DNA’s structure suggests a mechanism for replication. Each strand contains the information necessary to reconstruct each other. But how? After DNA was determined to be the hereditary material, the next question was, how DNA was replicated? The strands are said to be complimentary to each other. That is, DNA’s structure suggests a mechanism for replication. Each strand contains the information necessary to reconstruct each other. But how?

3. Watson and Crick Watson and Crick proposed a semiconservative model in which the new DNA strand formed contained 1/2 of the original DNA and 1/2 newly synthesized DNA--one strand was original and one strand was new. They couldn’t rule out a model where somehow the old DNA stayed together and the newly synthesized DNA strand was completely new. Watson and Crick proposed a semiconservative model in which the new DNA strand formed contained 1/2 of the original DNA and 1/2 newly synthesized DNA--one strand was original and one strand was new. They couldn’t rule out a model where somehow the old DNA stayed together and the newly synthesized DNA strand was completely new.

4. Watson and Crick Additionally, they could not rule out a dispersive model where both strands of DNA consisted of old and new DNA. The mechanisms for these three models were difficult to elucidate but Matthew Meselson and Franklin Stahl developed experiments to test them. Additionally, they could not rule out a dispersive model where both strands of DNA consisted of old and new DNA. The mechanisms for these three models were difficult to elucidate but Matthew Meselson and Franklin Stahl developed experiments to test them.

5. The Meselson-Stahl Experiment E. coli cells were cultured in a medium containing heavy nitrogen, 15 N. After several generations the bacteria were transferred into a medium containing normal nitrogen, 14 N. Ideally, all DNA synthesized the first time through contained heavy nitrogen. All DNA synthesized in the normal nitrogen tube (after the transfer) would be lighter than the DNA from the original culture. E. coli cells were cultured in a medium containing heavy nitrogen, 15 N. After several generations the bacteria were transferred into a medium containing normal nitrogen, 14 N. Ideally, all DNA synthesized the first time through contained heavy nitrogen. All DNA synthesized in the normal nitrogen tube (after the transfer) would be lighter than the DNA from the original culture.

6. The Meselson-Stahl Experiment Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If replication followed the conservative model, the first replication would contain 2 bands of DNA, one light and one heavy; and the 2nd replication would show the same thing (2 bands). Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If replication followed the conservative model, the first replication would contain 2 bands of DNA, one light and one heavy; and the 2nd replication would show the same thing (2 bands).

7. The Meselson-Stahl Experiment Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If it followed the dispersive model, one band would be seen containing hybrid DNA. This was seen after the first replication, but not the second*. Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If it followed the dispersive model, one band would be seen containing hybrid DNA. This was seen after the first replication, but not the second*.

8. The Meselson-Stahl Experiment Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If it followed the semiconservative model one band would be seen after the first replication and 2 would be seen after the 2nd replication. Their hypotheses: (what would be seen after centrifuging the tubes containing DNA) If it followed the semiconservative model one band would be seen after the first replication and 2 would be seen after the 2nd replication.

10. The Meselson-Stahl Experiment After they transferred the DNA from the 15 N tube to the 14 N tube, they waited 20 minutes (1 replication) and then centrifuged the tube. They were able to detect one band in the centrifuge tube. In a separate tube, they waited for 2 rounds of replication (40 min.) and were able to detect 2 bands in the centrifuge tube. After they transferred the DNA from the 15 N tube to the 14 N tube, they waited 20 minutes (1 replication) and then centrifuged the tube. They were able to detect one band in the centrifuge tube. In a separate tube, they waited for 2 rounds of replication (40 min.) and were able to detect 2 bands in the centrifuge tube.

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13. The Meselson-Stahl Experiment Conclusion: The semiconservative model was followed. A light band was detected following the 1st replication and 2 bands, (one light and one heavy) were detected following the 2nd replication. Conclusion: The semiconservative model was followed. A light band was detected following the 1st replication and 2 bands, (one light and one heavy) were detected following the 2nd replication.

14. Thus, it was determined that due to the complementary base pairing between the two strands, each one contains the information necessary to construct the other. Take a moment and show this in your notes using this sequence: 5’-A-C-T-C-G-G-T-A-A-3’ & 3’-T-G-A-G-C-C-A-T-T-5’ Thus, it was determined that due to the complementary base pairing between the two strands, each one contains the information necessary to construct the other. Take a moment and show this in your notes using this sequence: 5’-A-C-T-C-G-G-T-A-A-3’ & 3’-T-G-A-G-C-C-A-T-T-5’ DNA Replication

15. So once the mechanism for DNA replication was worked out, the details for how it occurred became the topic of interest. DNA Replication

16. DNA replication begins at a site called the “origin of replication.” Prokaryotes have one origin of replication. Eukaryotes have hundreds of thousands of origins of replication. DNA replication begins at a site called the “origin of replication.” Prokaryotes have one origin of replication. Eukaryotes have hundreds of thousands of origins of replication.

17. DNA Replication Here is an electron micrograph and a schematic representation of bacterial DNA replication.

18. Recall, John Cairns did a lot of elegant experiments using radioactive tracers to show how DNA was replicated (semi- conservative), and to show the length of the DNA found within cells. DNA Replication http://schaechter.asmblog.org/schaechter/2013/03/pictures-considered-the-e-coli-chromosome-caught-in-the-act-of-replicating.html http://schaechter.asmblog.org/schaechter/2015/01/pictures-considered-23-what-grains-tell.html

19. 19 There is a complex system of enzymes and other proteins involved in DNA replication: Helicase DNA polymerase Topoisomerase/DNA gyrase Primase DNA Ligase Single Strand Binding Proteins There is a complex system of enzymes and other proteins involved in DNA replication: Helicase DNA polymerase Topoisomerase/DNA gyrase Primase DNA Ligase Single Strand Binding Proteins 19 DNA Replication

20. Helicase is the enzyme responsible for untwisting the double helix at the replication fork. This separates the parental strands of DNA making them available for use as template strands. Helicase is the enzyme responsible for untwisting the double helix at the replication fork. This separates the parental strands of DNA making them available for use as template strands.

21. DNA Replication This untwisting actually causes a greater amount of twisting ahead of the replication fork, and an enzyme called topoisomerase helps to reduce this twisting.

22. DNA Replication After helicase separates the two parental strands, single strand binding protein binds to the DNA strands stabilizing them until new DNA synthesis occurs.

23. DNA Replication Primase works to join RNA nucleotides together creating primers complementary to the DNA template strand. This is the site of initiation, and new DNA strands will be synthesized here. Primase works to join RNA nucleotides together creating primers complementary to the DNA template strand. This is the site of initiation, and new DNA strands will be synthesized here.

24. DNA Replication Primers are the short nucleotide fragments (DNA or RNA) with an available free 3’ end to which DNA polymerase III (DNA pol III) will add nucleotides according to the base paring rules. Primase is the enzyme that starts an RNA chain from scratch creating a primer that can initiate the synthesis of a new DNA strand. Primers are the short nucleotide fragments (DNA or RNA) with an available free 3’ end to which DNA polymerase III (DNA pol III) will add nucleotides according to the base paring rules. Primase is the enzyme that starts an RNA chain from scratch creating a primer that can initiate the synthesis of a new DNA strand.

25. DNA Replication DNA pol I functions to replace the RNA nucleotide primers with DNA. All of the nucleotides of the RNA primer are eventually replaced by DNA pol I forming a continuous DNA molecule. DNA ligase closes the gap left between the last two nucleotides joining all of the Okazaki fragments into one continuous strand. DNA pol I functions to replace the RNA nucleotide primers with DNA. All of the nucleotides of the RNA primer are eventually replaced by DNA pol I forming a continuous DNA molecule. DNA ligase closes the gap left between the last two nucleotides joining all of the Okazaki fragments into one continuous strand.

26. DNA Replication Keep in mind that all of the molecules described in the replication process actually work together to maximize DNA production. Think of them as the individual parts of a “DNA synthesis machine.” Keep in mind that all of the molecules described in the replication process actually work together to maximize DNA production. Think of them as the individual parts of a “DNA synthesis machine.”

27. DNA Replication The leading strand of DNA synthesis only needs one primer. The lagging strand needs a new primer for each Okazaki fragment added to the growing strand. The leading strand of DNA synthesis only needs one primer. The lagging strand needs a new primer for each Okazaki fragment added to the growing strand.

28. DNA Replication DNA polymerases are enzymes that catalyze the elongation of DNA at the replication fork. One by one, nucleotides are added by DNA polymerase to the growing end of the DNA strand--the free 3’ end. DNA always grows 5’-- >3’ adding to the free 3’ end. DNA polymerases are enzymes that catalyze the elongation of DNA at the replication fork. One by one, nucleotides are added by DNA polymerase to the growing end of the DNA strand--the free 3’ end. DNA always grows 5’-- >3’ adding to the free 3’ end.

30. DNA Replication The 2 strands of the DNA are antiparallel, they are oriented in opposite directions. This poses replication problems. DNA polymerase only adds nucleotides to the free 3’ end. Thus, it can only elongate in the 5’-->3’ direction. You get what is known as a leading strand and a lagging strand. The 2 strands of the DNA are antiparallel, they are oriented in opposite directions. This poses replication problems. DNA polymerase only adds nucleotides to the free 3’ end. Thus, it can only elongate in the 5’-->3’ direction. You get what is known as a leading strand and a lagging strand.

31. DNA Replication Along the leading strand, DNA pol III synthesizes a complementary strand continuously in the 5’-->3’ direction.

32. DNA Replication Along what is known as the lagging strand, DNA pol III synthesizes the new DNA strand in the 5’-->3’ direction in a series of segments known as Okazaki fragments which form as the replication fork opens up.

33. DNA Replication The opening of the fork allows the DNA pol III to hop from one fragment to the next on the lagging strand template and synthesize more DNA. These fragments are spliced together by DNA ligase forming a single strand. The opening of the fork allows the DNA pol III to hop from one fragment to the next on the lagging strand template and synthesize more DNA. These fragments are spliced together by DNA ligase forming a single strand.

35. DNA Replication DNA Replication Video

36. DNA Replication The main importance of replicating the DNA is the ability to do it without error. Errors in completed eukaryotic DNA occur in approximately 1 in 10 billion nucleotides. Initial errors occur at a rate of about 1 in 100,000. Proofreading mechanisms by DNA polymerase fix many of the problems. The main importance of replicating the DNA is the ability to do it without error. Errors in completed eukaryotic DNA occur in approximately 1 in 10 billion nucleotides. Initial errors occur at a rate of about 1 in 100,000. Proofreading mechanisms by DNA polymerase fix many of the problems.

37. DNA Replication If an error escapes proofreading, they are often fixed by special enzymes within the cell-- but even these are not 100% effective at removing all errors. Additionally, some errors occur after DNA synthesis has been completed. If an error escapes proofreading, they are often fixed by special enzymes within the cell-- but even these are not 100% effective at removing all errors. Additionally, some errors occur after DNA synthesis has been completed.

38. Excision Repair Errors that occur as a result of the environment (radiation, chemicals, X-rays, etc.) can often be fixed by DNA polymerase and ligase. This is a way the cell tries (usually effectively) to fix problems before they get perpetuated (cancer). Errors that occur as a result of the environment (radiation, chemicals, X-rays, etc.) can often be fixed by DNA polymerase and ligase. This is a way the cell tries (usually effectively) to fix problems before they get perpetuated (cancer).