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.

11

Chapter 16Chapter 16

The Molecular Basis of Inheritance

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

Watson and CrickWatson 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?


• 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.


• 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.

The Meselson-Stahl Experiment


• E. coli cells were cultured in a medium containing heavy nitrogen, 15N.

• After several generations the bacteria were transferred into a medium containing normal nitrogen, 14N.

• 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, 15N.

• After several generations the bacteria were transferred into a medium containing normal nitrogen, 14N.

• 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.



• 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).



• 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*.



• 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.



• After they transferred the DNA from the 15N tube to the 14N 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 15N tube to the 14N 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.

1212



• 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.

• 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 ReplicationDNA Replication

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

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



• 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.


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

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

• 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.

• 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.


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

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

1919

• 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



• 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.


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

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


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

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


• 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.


• 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.


• 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.


• 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.”

DNA ReplicationDNA 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.

DNA ReplicationDNA 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.

DNA ReplicationDNA 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.


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

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


• 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.

• 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.

DNA ReplicationDNA 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.


• DNA Replication Video• DNA Replication Video

http://bcs.whfreeman.com/thelifewire/content/chp11/1103s.swf

http://bcs.whfreeman.com/thelifewire/content/chp11/1103s.swf


• 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.

DNA ReplicationDNA 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.

Excision RepairExcision 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).

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.

Documents

bands of dna

original dna

strands of dna

old dna

crickafter dna

dna replicat

hybrid dna

new dna strand