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A diagram explaining the Meselson Stahl experiment. In the first part of the experiment DNA is replicated in the presence of heavy 15N medium. This produces all heavy DNA strands. Next they moved the cells to light 14N medium. If DNA was replicated conservatively, one would expect to see one heavy band and one light band. However, they saw only a medium size band. This is consistent with semiconservative and dispersive replication. Finally, they allowed the bacteria to undergo another round of replication in the light medium. If DNA was replicated dispersively, one would expect only a medium size band. However, they saw a medium band and a light band. The only mechanism that explains these results is semi-conservative replication.
Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in 14 N. DNA grown in 15 N (blue band) was heavier than DNA grown in 14 N (red band), and sedimented to a lower level on ultracentrifugation. After one round of replication, the DNA sedimented halfway between the 15 N and 14 N levels (purple band), ruling out the conservative model of replication. After a second round of replication, the dispersive model of replication was ruled out. These data supported the semiconservative replication model.
  • What would have been the conclusion of Meselson and Stahl’s experiment if, after the first generation, they had found two bands of DNA?

Dna replication in bacteria

DNA replication has been well studied in bacteria primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs (Mbp) in a single circular chromosome and all of it is replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle bidirectionally (i.e., in both directions). This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs with few errors.

DNA replication uses a large number of proteins and enzymes ( [link] ). One of the key players is the enzyme DNA polymerase , also known as DNA pol. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. DNA pol III adds deoxyribonucleotides each complementary to a nucleotide on the template strand, one by one to the 3’-OH group of the growing DNA chain. The addition of these nucleotides requires energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide (a triphosphate nucleotide), similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP) ( [link] ). When the bond between the phosphates is broken and diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis between the incoming nucleotide and the free 3’-OH group on the growing DNA strand.

Diagram of dGTP. In the center is deoxyribose which is a pentagon shaped sugar. The top point has an oxygen. Then, moving around the shape are carbons 1, 2, 3, and 4; carbon 5 is attached to carbon 4 but not in the ring. Attached to carbon 1 is a structure made of 2 carbon and nitrogen rings bound along their ends; this is guanine. Carbon 2 has only Hs attached to it. Carbon 3 has an H and an OH. Carbon 4 has an N and Carbon 5. Carbon 5 is attached to 3 phosphate groups in a row (labeled triphosphate). Each phosphate group is made of phosphorus attached to 4 oxygen atoms.
This structure shows the guanosine triphosphate deoxyribonucleotide that is incorporated into a growing DNA strand by cleaving the two end phosphate groups from the molecule and transferring the energy to the sugar phosphate bond. The other three nucleotides form analogous structures.

Initiation

The initiation of replication occurs at specific nucleotide sequence called the origin of replication , where various proteins bind to begin the replication process. E. coli has a single origin of replication (as do most prokaryotes), called oriC , on its one chromosome. The origin of replication is approximately 245 base pairs long and is rich in adenine-thymine (AT) sequences.

Some of the proteins that bind to the origin of replication are important in making single-stranded regions of DNA accessible for replication. Chromosomal DNA is typically wrapped around histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is supercoiled , or extensively wrapped and twisted on itself. This packaging makes the information in the DNA molecule inaccessible. However, enzymes called topoisomerases change the shape and supercoiling of the chromosome. For bacterial DNA replication to begin, the supercoiled chromosome is relaxed by topoisomerase II , also called DNA gyrase . An enzyme called helicase then separates the DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs. Recall that AT sequences have fewer hydrogen bonds and, hence, have weaker interactions than guanine-cytosine (GC) sequences. These enzymes require ATP hydrolysis. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication, allowing for bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope; as a result, this structure is called a replication bubble . The DNA near each replication fork is coated with single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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