DNA Replication

DNA Replication is the process of creating two identical DNA molecules from an original parent DNA molecule. This step occurs in late Interphase before the start of Mitosis, when a cell will need two sets of DNA for each of the Daughter Cells produced.

This process needs a Template Strand (an existing DNA strand), and produces an exact replica. This process differs slightly between Prokaryotes and Eukaryotes.

To get a full grasp of DNA Replication, we must look at the structure of DNA. DNA is composed of nucleotides comprised of a Nitrogenous Base (A, T, G, C), Deoxyribose, and a phosphates. The nucleotides are bound together in two strands, and the strands run Antiparallel to eachother (opposite directions). One strand will end with a phosphate (called the 5' end), while the other will end with a deoxyribose (the 3' end). The nitrogenous bases are connected together by Hydrogen Bonds and always pair as follows: A-T and G-C

If replication had been conservative, then Generation I would have shown two bands!

DNA follows a Semiconservative Model, where each new double strand is composed of one of the template strands and one new strand.

This was confirmed by Meselson and Stahl in 1958 in the following experiment:

Labelled DNA in E. coli with ‘heavy’ nitrogen (¹⁵N). Then allowed it to replicate 1 or 2 times in a nutrient solution containing ‘light’ nitrogen (¹⁴N).
DNA composed of ONLY ¹⁵N is very dense, DNA made of half ¹⁵N and half ¹⁴N is somewhat dense, while DNA composed only of ¹⁴N is least dense.

Steps in DNA Replication

A. DNA Separation

1. DNA is unravelled (H-bonds broken) at the replication origin by the enzyme DNA Helicase, creating a Replication Fork.

2. Each parent strand (the 5’ to 3’ strand and the 3’ to 5’ strand) is now exposed and can be replicated by the insertion of free-floating nucleotides in the nucleoplasm.

3. As DNA uncoils, it creates tension that can lead to twisting as seen in phone cords.

This is prevented by topoisomerase enzymes.

4. DNA is prevented from reannealing by single-stranded binding (SSB) proteins.

5. DNA separates at the replication fork in both directions, creating a replication bubble.

B. DNA Replication

6. Exposed DNA is replicated by the enzyme DNA polymerase. This enzyme adds a nucleotide to the 3’ end of the new strand.

Note: the DNA is attached as Deoxyribonucleoside Triphosphates (nucleic acids with 3 phosphate groups)
Two phosphates are dropped from each nucleotide, providing bonding energy to build the strand (see p.285)

7. For DNA polymerase to work, there must be an available 3’ nucleotide. This is provided by the enzyme RNA primase which adds a short complementary strand of RNA called the RNA primer. The primer provides the free 3’ end for DNA to be added to.

8. DNA polymerase III grows the DNA strand continuously as the replication fork continues to open.

9. DNA replication proceeds smoothly on the 3’ to 5’ parent strand. This new strand is called the leading strand.

10. The 5’ to 3’ parent strand builds short stretches of DNA called Okazaki fragments or the lagging strand.

11. DNA polymerase I removes the primer and the final phosphodiester bond to join the two Okazaki fragments is achieved using DNA ligase.

C. Proofread and Repair

12. DNA polymerase II replaces incorrect nucleotides and inserts the correct nucleotide (If mistakes are made, mutations can occur!)

Telomeres

The last stretch of the lagging strand of DNA cannot be finished because once the RNA primer falls off it can’t be replaced with DNA, so with each replication, the DNA strand gets shorter and shorter.

To counter this the ends of chromosomes have stretches of non-coding DNA called telomeres, these can be lost without harm to the cell. Also read p. 295-298 to learn the importance (or lack thereof) of telomeres to disease.

Textbook Practice

p.282-290# 1-4, 6, 7

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