1. 01 Introduction to Cell Respiration STUDENT HANDOUTS
DNA Part II:
The "Stuff" of Life
Its Replication & Its Unending Repair

2. Replication of DNA
In their Nobel Prize winning paper on the structure of DNA, Watson and Crick stated that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.“ They later published a second paper which suggested a hypothesis for the replication of DNA. That each strand of the DNA molecule could act as a template for the synthesis of opposite strand of the DNA molecule.

3. Possible Scenarios for DNA Replication
THREE possible scenarios for DNA replication
Conservative DNA replication- the original helix is kept intact, giving rise to a completely new helix.
Semi-conservative replication- the 2 strands separate and each serves as a template that gives rise to 2 helices which are composed of one side being an original strand and the other side being a new strand that is complementary to the original strand.
Dispersive replication- the replicated helices are composed of both a new strand and pieces of the original strand.

4. Experiment of Meselson and Stahl
Matt Meselson (1930- ) and Frank Stahl (1929- ) in 1958 demonstrated that replication was indeed semi-conservative using radioactive nucleotides and dividing bacteria.
Matt Meselson (left) and Frank Stahl (right) in 1958 demonstrated that replication was semi-conservative using radioactive nucleotides with dividing bacteria supporting Watson’s and Crick’s hypothesis

5. Proof for DNA Replication
The bacteria was cultured in a medium containing 15N so that all the bacterial DNA was labeled with “heavy” nitrogen. Ask students why we refer to it as “heavy”? (Because the stable isotope of nitrogen has a molar mass of 14, not 15! Therefore this DNA is heavier.) Ask students to examine the experimental design presented on this slide. What is the significance of “heavy nitrogen” as it relates to the design? (Upon subsequent centrifuging, the “heavy” DNA can be extracted from the bottom layer of the centrifuge tube.)
Next, the bacteria are transferred to medium containing 14N and allowed to replicate once. The DNA was extracted from the bacteria and was centrifuged in cesium chloride.
The experiment was repeated but this time the bacteria were allowed to replicate twice.
It was known the heavier DNA (radioactive) migrated to the bottom of the cesium chloride when centrifuged, and that the DNA that had only nonradioactive nucleotides would be closer to the top of the tube. DNA that had both radioactive and nonradioactive would migrate between the two radioactive and nonradioactive DNA.
After one replication, the conservative scenario was eliminated as only one banding pattern appeared at the position for DNA that contained radioactive and nonradioactive nucleotides. However this did not differentiate between dispersive or semiconservative.
Allowing the bacteria to replicate twice did differentiate between the semi conservative scenario and the dispersive scenario. The fact that two bands appeared instead of one appeared, supported the scenario for semiconservative replication.

6. Simple Model Proposed for DNA Replication

7. Prokaryotic DNA Replication
The prokaryotic chromosome attaches to the plasma membrane. The DNA is then replicated in both directions.
Emphasize that the chromosome of a prokaryotic cell is circular as opposed to linear as in eukaryotic cells. Only one origin of replication which attaches to the plasma membrane is present in prokaryotic cells. Replication of the prokaryotic chromosome occurs in both directions as does replication of eukaryotic chromosomes. Emphasize that prokaryotes have far fewer DNA base pairs than eukaryotes. E. coli has about 4.6 million base pairs whereas a human eukaryotic cell has 3 billion base pairs to replicate.

8. Eukaryotic DNA Replication
DNA replication occurs simultaneously in many locations along the very long eukaryotic chromosomes.
As stated previously, DNA replication occurs in both directions within eukaryotic cells. The site where it begins is called the origin of replication.
Depending on the source, it has been reported that DNA elongation occurs at a rate of nucleotides added per second in eukaryotic cells.
Note in E. coli there are 4.6 million base pairs in its single, circular chromosome and only one origin of replication. In human somatic cells, there are three billion base pairs, which is FAR more than present in prokaryotic cells.
It would take too long to have only one origin of replication in replicating the DNA. It is estimated that there are 5 × 104 replication origins among a human’s 23 chromosomes containing 3 × 109 base pairs.
Three replication bubbles are visible along the DNA within this cultured Chinese hamster cell. The arrows indicate the directions of DNA replication at the two ends of the bubble.

9. Helicases
Helicases are enzymes responsible for the unwinding of the DNA molecule. They unwind the DNA in both directions
Historical note: DNA helicases were first discovered in E. coli in The discoverers of this helicase described the molecule as a “DNA unwinding enzyme” that is “found to denature (fancy word for “break” the H-bonds) DNA duplexes in an ATP-dependent reaction, without detectably degrading”. The first eukaryotic DNA helicase discovery was in 1978 and found in the lily plant.
DNA helicases are essential during DNA replication because they separate double-stranded DNA into single strands allowing each strand to be copied.  The helicases form replication forks as the helicases unwind the DNA in both directions. Students are responsible for knowing the function of helicase and its role in DNA replication.

10. Releasing Stress in the DNA Molecule
Since DNA is a double helix, there will be tension in the DNA strand that causes it to tangle as it is unwound by the helicase. The enzymes topoisomerase I and II are responsible for relieving that stress by clipping one or two strands of the DNA.

11. Adding Nucleotides as Triphosphates
Nucleotides are always added on as triphosphates. When the nucleotides are added then two phosphates are cleaved off making a pyrophosphate.

12. Synthesis ALWAYS occurs in the 5 to 3 direction!
Students are responsible for knowing the function of DNA polymerase and its role in DNA replication.
Emphasize the following:
DNA polymerase is the enzyme responsible for base pairing the correct nucleotide to the template. It is facilitates the formation of the H-bonds that make the “rungs” of the DNA ladder. The base pairing is:
Adenine with thymine
Guanine with cytosine
Nucleotides are added as triphosphates with two phosphates (pyrophosphates cleaved off).
Nucleotides can only be added to the 3’ end.
DNA synthesis (grows in length) is ALWAYS from 5’ to 3’.
When a DNA molecule is being synthesized , the nucleotides are added as triphosphates, and two phosphates are removed. Nucleotides are always added to the 3 end!

13. In Need of a Primer!
DNA polymerase must always attach the com-plementary nucleotide to a 3 end of the deoxyribose sugar molecule. So, in the very beginning a small RNA primer must be laid down in order to start the process of DNA replication. Primase is the enzyme responsible for this.
Students are not responsible for knowing the name of the enzyme primase but are responsible for knowing about the process and the fact that DNA polymerase must always attach new nucleotides to the 3’ end of the sugar with covalent bonds on the previous nucleotide of that strand.
DNA Polymerase III is the polymerase that does the “normal” base pairing (H-bonds) during replication. DNA polymerase I is the enzyme that replaces the RNA nucleotides of the primer with DNA nucleotides. However, students simply need to know the term DNA Polymerase and will not be expected to distinguish between I and III.

14. Putting Down a RNA Primer
The RNA nucleotide primers will be replaced with DNA nucleotides by another enzyme, DNA polymerase.
Emphasize that each side of the DNA molecule is a template for the newly synthesized strand.
Ask students if they see any problem with this process! Hopefully they notice that only one side of the DNA molecule can be replicated continuously in one long strand (called the leading strand) due to the fact that replication must always proceed from 5’ to 3’ direction. What about the other side? Never fear! That is addressed in the next slide.
RNA nucleotides (red pentagons) are being laid down by primase before DNA polymerase begins DNA replication.

15. The Lagging Strand
DNA polymerase can only add to the 3’ end of the deoxyribose sugar. That being the case, synthesis on the opposite side must wait until enough room has opened up in the bubble and synthesize in the opposite direction forming fragments of DNA called Okazaki fragments. As one fragment lengthens and “runs into” the other the RNA primers are replaced with appropriate DNA base pairs and DNA ligase “ties” the fragments together. This strand is called the lagging strand. Apt, don’t you think?
Emphasize that both DNA polymerase and ligase facilitate the formation of COVALENT (phosphodiester) bonds.

16. The Lagging Strand and Ligase
Students are responsible for knowing ligase and its role in DNA replication.
Approach this example with caution, but if student’s know the name of the procedure when a female human has her “tubes tied” formally named a “tubal ligation”, it helps them remember both the name and function of this ligase enzyme as it relates to DNA synthesis.
This animation, shows the leading strand being synthesized followed by the lagging strand. The enzyme named ligase ties them together.

17. Function of Telomeres
Once DNA has been replicated, there is one problem. The usual replication machinery provides no way to complete the 5 ends after the RNA primer is removed, so repeated rounds of replication produce shorter and shorter DNA molecules.
To compensate for this repeated shortening process, repetitive sequences of DNA are added. These are noncoding sequences and called telomeres.
Students are not responsible for this information but is important in explaining why chromosomes do get shorter and shorter every time they replicate
Challenge students to explain why the situation in the slide presents a problem. (After the RNA primer is removed from the end of the newly synthesized strand, it is not possible to create a double stranded section in front of this region from which DNA polymerase can then work to replicate the 5’ end).
Telomeres are repeated sequences of TTAGGG (in humans) on one strand of DNA bound to AATCCC on the other strand. Telomeres have been compared with the plastic tips on shoelaces because they prevent chromosome ends from fraying and sticking to each other, which would scramble an organism's genetic information and could lead to cancer, other diseases or death.
Yet, each time a cell divides, the telomeres get shorter. When they get too short, the cell no longer can divide and becomes inactive or "senescent" or dies. This process is associated with aging. In human blood cells, the length of telomeres ranges from 8,000 base pairs at birth to 3,000 base pairs as people age and as low as 1,500 in elderly people. (An entire chromosome has about 150 million base pairs.) Each time a cell divides, the average person loses 30 to 200 base pairs from the ends of that cell's telomeres.
Cells normally can divide only about 50 to 70 times, with telomeres getting progressively shorter until the cells become senescent, die or sustain genetic damage that can cause cancer.
Gametes, germ cells, epithelial cells and cancer cells are an exception to the rule, as explained in the next slide.

18. Maintaining Telomere Length in Gametes and Germ Cells
In gametes, the shortening of telomeres would cause serious problems. If chromosomes of gametes became shorter each time during replication, then essential genes would eventually be missing. An enzyme complex called telomerase catalyzes the lengthening of telomeres in gametes.
Telomerase is a ribonucleoprotein that is an enzyme which adds DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. This region of repeated nucleotide called telomeres contains noncoding DNA and hinders the loss of important DNA from chromosome ends. As a result, every time the chromosome is copied only 100–200 nucleotides are lost, which causes no damage to the organism's DNA. Telomerase is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle.
Telomerase is also active in germ cells, blood cells, epithelial cells and cancer cells (which is why they seem immortal). All yeast cells have telomerase as these cells are single celled organisms that replicate. In humans most somatic cells do not have active telomerase.
Telomerase is an enzyme complex as it also contains the RNA primer for the DNA telomere.

19. DNA Repair
Errors in DNA replication occur about 1 in every 10,000 base pairs. Not bad, but with 6 billion bases being replicated that amounts to 60,000 mistakes every time a cell divides.
DNA repair systems repair about 99% of these mistakes.
Proofreading and Repairing DNA
DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
In mismatch repair of DNA, repair enzymes correct errors in base pairing
DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example)
In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA.
Ask students why these errors are not catastrophic. Hopefully they remember a bit about protein synthesis and that there are often multiple codons coding for the same amino acid so there is forgiveness in the process!

20. 01 Introduction to Cell Respiration STUDENT HANDOUTS
Created by:
Carol Leibl
Director of Science Programs
The National Math and Science Initiative
Dallas, TX