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68 Basics of DNA Replication

DNA Structure and Function

Learning Objectives

By the end of this section, you will be able to do the following:

  • Explain how the structure of DNA reveals the replication process
  • Describe the Meselson and Stahl experiments

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. In their 1953 paper, Watson and Crick penned an incredible understatement: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” With specific base pairs, the sequence of one DNA strand can be predicted from its complement. The double-helix model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested ((Figure)): conservative, semi-conservative, and dispersive.

The three suggested models of DNA replication. Gray indicates the original DNA strands, and blue indicates newly synthesized DNA.


Illustration shows the conservative, semi-conservative, and dispersive models of D N A synthesis. In the conservative model, the D N A is replicated and both newly synthesized strands are paired together. In the semi-conservative model, each newly synthesized strand pairs with a parent strand. In the dispersive model, newly synthesized D N A is interspersed with parent D N A within both D N A strands.

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N), which gets incorporated into nitrogenous bases, and eventually into the DNA ((Figure)).

Meselson and Stahl experimented with E. coli grown first in heavy nitrogen (15N) then in 14N. DNA grown in 15N (red band) is heavier than DNA grown in 14N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15N is switched to media containing 14N, after one round of cell division the DNA sediments halfway between the 15N and 14N levels, indicating that it now contains fifty percent 14N. In subsequent cell divisions, an increasing amount of DNA contains 14N only. These data support the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)


Illustration shows an experiment in which E coli was grown initially in media containing superscript 15 baseline upper case N  nucleotides. When the D N A was extracted and run in an ultracentrifuge, a band of D N A appeared low in the tube. The culture was next placed in the superscirpt 14 baseline upper case N medium. After one generation, all of the D N A appeared in the middle of the tube, indicating that the D N A was a mixture of half superscript 14 baseline upper N and half superscript 15 baseline upper N, D N A. After two generations, half of the D N A appeared in the middle of the tube, and half appeared higher up, indicating that half the D N A contained 50% superscript 15 baseline upper N, and half contained superscript 14 baseline upper N only. In subsequent generations, more and more of the D N A appeared in the upper, superscript 14 baseline upper N band.

The E. coli culture was then placed into medium containing 14N and allowed to grow for several generations. After each of the first few generations, the cells were harvested and the DNA was isolated, then centrifuged at high speeds in an ultracentrifuge. During the centrifugation, the DNA was loaded into a gradient (typically a solution of salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its buoyant density: the density within the gradient at which it floats. DNA grown in 15N will form a band at a higher density position (i.e., farther down the centrifuge tube) than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N after they had been shifted from 15N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N and 14N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. And for this reason, therefore, the other two models were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strands will be complementary to the parental or “old” strands. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Section Summary

During cell division, each daughter cell receives a copy of each molecule of DNA by a process known as DNA replication. The single chromosome of a prokaryote or each chromosome of a eukaryote consists of a single continuous double helix. The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In the conservative model of replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative model suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized; after replication, each double-stranded DNA retains the parental or “old” strand and one “new” strand. The dispersive model suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA. The Meselson and Stahl experiment supported the semi-conservative model of replication, in which an entire replicated chromosome consists of one parental strand and one newly synthesized strand of DNA.

Review Questions

Meselson and Stahl’s experiments proved that DNA replicates by which mode?

  1. conservative
  2. semi-conservative
  3. dispersive
  4. none of the above

If the sequence of the 5′-3′ strand is AATGCTAC, then the complementary sequence has the following sequence:

  1. 3′-AATGCTAC-5′
  2. 3′-CATCGTAA-5′
  3. 3′-TTACGATG-5′
  4. 3′-GTAGCATT-5′

How did Meselson and Stahl support Watson and Crick’s double-helix model?

  1. They demonstrated that each strand serves as a template for synthesizing a new strand of DNA.
  2. They showed that the DNA strands break and recombine without losing genetic material.
  3. They proved that DNA maintains a double-helix structure while undergoing semi-conservative replication.
  4. They demonstrated that conservative replication maintains the complementary base pairing of each DNA helix.

Critical Thinking Questions

How did the scientific community learn that DNA replication takes place in a semi-conservative fashion?

Meselson’s experiments with E. coli grown in 15N deduced this finding.

Imagine the Meselson and Stahl experiments had supported conservative replication instead of semi-conservative replication. What results would you predict to observe after two rounds of replication? Be specific regarding percent distributions of DNA incorporating 15N and 14N in the gradient.

Following two rounds of conservative replication, two bands would be detected after ultracentrifugation. A lower (heavier) band would be at the 15N density, and would comprise 25% of the total DNA. A second, higher (lighter) band would be at the 14N density, and would contain 75% of the total DNA.

Sours: https://opentextbc.ca/biology2eopenstax/chapter/basics-of-dna-replication/

iGen3 03-01

Semi-Conservative, Conservative, & Dispersive models of DNA replication

In the semi-conservative model, the two parental strands separate and each makes a copy of itself. After one round of replication, the two daughter molecules each comprises one old and one new strand. Note that after two rounds, two of the DNA molecules consist only of new material, while the other two contain one old and one new strand.

In the conservative model, the parental molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. This is repeated in the second round.

In the dispersive model, material in the two parental strands is distributed more or less randomly between two daughter molecules. In the model shown here, old material is distributed symmetrically between the two daughters molecules. Other distributions are possible.

The semi-conservative model is the intuitively appealing model, because separation of the two strands provides two templates, each of which carries all the information of the original molecule. It also turns out to be the correct one (Meselson & Stahl 1958).


Figure © 2010 PJ Russell, iGenetics 3rd ed.; all text material © 2011 by Steven M. Carr

Sours: https://www.mun.ca/biology/scarr/iGen3_03-01.html
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7.3A: Basics of DNA Replication

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DNA replication uses a semi-conservative method that results in a double-stranded DNA with one parental strand and a new daughter strand.

LEARNING OBJECTIVES

Explain how the Meselson and Stahl experiment conclusively established that DNA replication is semi-conservative.

Key Takeaways

Key Points

  • There were three models suggested for DNA replication: conservative, semi-conservative, and dispersive.
  • The conservative method of replication suggests that parental DNA remains together and newly-formed daughter strands are also together.
  • The semi-conservative method of replication suggests that the two parental DNA strands serve as a template for new DNA and after replication, each double-stranded DNA contains one strand from the parental DNA and one new (daughter) strand.
  • The dispersive method of replication suggests that, after replication, the two daughter DNAs have alternating segments of both parental and newly-synthesized DNA interspersed on both strands.
  • Meselson and Stahl, using E. coli DNA made with two nitrogen istopes (14N and 15N) and density gradient centrifugation, determined that DNA replicated via the semi-conservative method of replication.

Key Terms

  • DNA replication: a biological process occuring in all living organisms that is the basis for biological inheritance
  • isotope: any of two or more forms of an element where the atoms have the same number of protons, but a different number of neutrons within their nuclei

Basics of DNA Replication

Watson and Crick’s discovery that DNA was a two-stranded double helix provided a hint as to how DNA is replicated. During cell division, each DNA molecule has to be perfectly copied to ensure identical DNA molecules to move to each of the two daughter cells. The double-stranded structure of DNA suggested that the two strands might separate during replication with each strand serving as a template from which the new complementary strand for each is copied, generating two double-stranded molecules from one.

Models of Replication

There were three models of replication possible from such a scheme: conservative, semi-conservative, and dispersive. In conservative replication, the two original DNA strands, known as the parental strands, would re-basepair with each other after being used as templates to synthesize new strands; and the two newly-synthesized strands, known as the daughter strands, would also basepair with each other; one of the two DNA molecules after replication would be “all-old” and the other would be “all-new”. In semi-conservative replication, each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand just synthesized, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand. In dispersive replication, after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands.

image

To determine which model of replication was accurate, a seminal experiment was performed in 1958 by two researchers: Matthew Meselson and Franklin Stahl.

Meselson and Stahl

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that is incorporated into nitrogenous bases and, eventually, into the DNA. The E. coliculture was then shifted into medium containing the common “light” isotope of nitrogen (14N) and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge in a tube in which a cesium chloride density gradient had been established. Some cells were allowed to grow for one more life cycle in 14N and spun again.

image

During the density gradient ultracentrifugation, the DNA was loaded into a gradient (Meselson and Stahl used a gradient of cesium chloride salt, although other materials such as sucrose can also be used to create a gradient) and spun at high speeds of 50,000 to 60,000 rpm. In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the cesium chloride solution being more dense the farther down the tube you went. Under these circumstances, during the spin the DNA was pulled down the ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient where the DNA molecules’ density matched that of the surrounding salt solution. At the point, the molecules stopped sedimenting and formed a stable band. By looking at the relative positions of bands of molecules run in the same gradients, you can determine the relative densities of different molecules. The molecules that form the lowest bands have the highest densities.

DNA from cells grown exclusively in 15N produced a lower band than DNA from cells grown exclusively in 14N. So DNA grown in 15N had a higher density, as would be expected of a molecule with a heavier isotope of nitrogen incorporated into its nitrogenous bases. Meselson and Stahl noted that after one generation of growth in 14N (after cells had been shifted from 15N), the DNA molecules produced only single band intermediate in position in between DNA of cells grown exclusively in 15N and DNA of cells grown exclusively in 14N. This suggested either a semi-conservative or dispersive mode of replication. Conservative replication would have resulted in two bands; one representing the parental DNA still with exclusively 15N in its nitrogenous bases and the other representing the daughter DNA with exclusively 14N in its nitrogenous bases. The single band actually seen indicated that all the DNA molecules contained equal amounts of both 15N and 14N.

The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N and the other corresponded to the band of exclusively 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Dispersive replication would have resulted in exclusively a single band in each new generation, with the band slowly moving up closer to the height of the 14N DNA band. Therefore, dispersive replication could also be ruled out.

Meselson and Stahl’s results established that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are synthesized. The new strand will be complementary to the parental or “old” strand and the new strand will remain basepaired to the old strand. So each “daughter” DNA actually consists of one “old” DNA strand and one newly-synthesized strand. When two daughter DNA copies are formed, they have the identical sequences to one another and identical sequences to the original parental DNA, and the two daughter DNAs are divided equally into the two daughter cells, producing daughter cells that are genetically identical to one another and genetically identical to the parent cell.

Sours: https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/7%3A_Microbial_Genetics/7.03%3A_DNA_Replication/7.3A%3A_Basics_of_DNA_Replication
DNA Synthesis
Matthew Meselson and Franklin Stahl were well acquainted with these three predictions, and they reasoned that if there were a way to distinguish old versus new DNA, it should be possible to test each prediction. Aware of previous studies that had relied on isotope labels as a way to differentiate between parental and progenymolecules, the scientists decided to see whether the same technique could be used to differentiate between parental and progeny DNA. If it could, Meselson and Stahl were hopeful that they would be able to determine which prediction and replication model was correct.

The duo thus began their experiment by choosing two isotopes of nitrogen—the common and lighter 14N, and the rare and heavier 15N (so-called "heavy" nitrogen)—as their labels and a technique known as cesium chloride (CsCl) equilibrium density gradient centrifugation as their sedimentation method. Meselson and Stahl opted for nitrogen because it is an essential chemical component of DNA; therefore, every time a cell divides and its DNA replicates, it incorporates new N atoms into the DNA of either one or both of its two daughter cells, depending on which model was correct. "If several different density species of DNA are present," they predicted, "each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way, DNA labeled with heavy nitrogen (15N) may be resolved from unlabeled DNA" (Meselson & Stahl, 1958).

The scientists then continued their experiment by growing a culture of E. colibacteria in a medium that had the heavier 15N (in the form of 15N-labeled ammonium chloride) as its only source of nitrogen. In fact, they did this for 14 bacterial generations, which was long enough to create a population of bacterial cells that contained only the heavier isotope (all the original 14N-containing cells had died by then). Next, they changed the medium to one containing only 14N-labeled ammonium salts as the sole nitrogen source. So, from that point onward, every new strand of DNA would be built with 14N rather than 15N.

Just prior to the addition of 14N and periodically thereafter, as the bacterial cells grew and replicated, Meselson and Stahl sampled DNA for use in equilibrium density gradient centrifugation to determine how much 15N (from the original or old DNA) versus 14N (from the new DNA) was present. For the centrifugation procedure, they mixed the DNA samples with a solution of cesium chloride and then centrifuged the samples for enough time to allow the heavier 15N and lighter 14N DNA to migrate to different positions in the centrifuge tube.

Sours: http://www.nature.com/scitable/topicpage/semi-conservative-dna-replication-meselson-and-stahl-421

Synthesis model dna

Learning Outcomes

  • Outline the basic steps in DNA replication
Illustration shows the conservative, semi-conservative, and dispersive models of DNA synthesis. In the conservative model, when DNA is replicated and both newly synthesized strands are paired together. In the semi-conservative model, each newly synthesized strand pairs with a parent strand. In the dispersive model, newly synthesized DNA is interspersed with parent DNA within both DNA strands.

Figure 1. The three suggested models of DNA replication. Grey indicates the original DNA strands, and blue indicates newly synthesized DNA.

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive (see Figure 1).

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 2).

Illustration shows an experiment in which E. coli was grown initially in media containing ^{15}N nucleotides. When the DNA was extracted and run in an ultracentrifuge, a band of DNA appeared low in the tube. The culture was next placed in ^{14}N medium. After one generation, all of the DNA appeared in the middle of the tube, indicating that the DNA was a mixture of half ^{14}N and half ^{15}N DNA. After two generations, half of the DNA appeared in the middle of the tube, and half appeared higher up, indicating that half the DNA contained 50% ^{15}N, and half contained ^{14}N only. In subsequent generations, more and more of the DNA appeared in the upper, ^{14}N band.

Figure 2. Meselson and Stahl experimented with E. coli grown first in heavy nitrogen (15N) then in 14N. DNA grown in 15N (red band) is heavier than DNA grown in 14N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15N is switched to media containing 14N, after one round of cell division the DNA sediments halfway between the 15N and 14N levels, indicating that it now contains fifty percent 14N. In subsequent cell divisions, an increasing amount of DNA contains 14N only. This data supports the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then shifted into medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15N will band at a higher density position than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N after they had been shifted from 15N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N and 14N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Click through this tutorial on DNA replication.

In Summary: Basics of DNA Replication

The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In conservative replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative method suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. The dispersive mode suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA. Experimental evidence showed DNA replication is semi-conservative.

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DNA replication in prokaryotic cell 3D animation with subtitle

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