See also DNA replication in bacteria
Arthur Kornberg by Allen Gathman, on Flickr
Arther Kornberg was born in 1918 in Brooklyn, NY. He earned a bachelor's degree in 1937 at the City College of New York and an M.D. at the University of Rochester in 1941. Kornberg first worked on enzymatic synthesis of coenzymes and inorganic phosphates. He then later turned to the biosynthesis of nucleic acids such as DNA. The types of proteins that do not catalyze formation of phosphodiester bonds were separated out by size, surface charge, etc. until DNA polymerase I, II, and III were isolated. DNA Polymerase enzyme is best known for its role in the replication of DNA. DNA polymerase catalyzes the polymerization of deoxyribonuceotides into a DNA strand. DNA Polymerase I and III are used during replication, while DNA Polymerase II is only used during repair. There are 5 known Prokaryotic DNA polymerases and 15 Eukaryotic DNA polymerases.
Kornberg needed three things in order to replicate DNA in a test tube that supposedly contained the enzyme responsible for DNA replication. The three things that are needed include a substrate, a DNA template, and some type of energy source. In order to replicate DNA begin by unwinding the double helical structure.
has oriC sequence that tells where to start unwinding.
DNA A binds to the oriC.
Eukaryotes have several origins of replication per chromosome, but prokaryotes only have one.
His findings from isolating DNA Polymerase in E. Coli won him a Nobel Prize in medicine in 1959.
Requirements for DNA polymerases
Requirements for DNA polymerases by Allen Gathman, on Flickr
Deoxyribonucleotide triphosphates, or dNTP, is a general term that represents the four deoxyribonucleotides that make up DNA. These dNTP's include dATP, dTTP, dGTP, and dCTP.
The removal of phosphates from the ATP molecule is a -Δ G reaction, and energy is released.
The dNTP binds to the 3' end and the two extra phosphates break off by the process of hydrolysis.
The right side is a template, a single strand of DNA. Also a portion of the strand must be double stranded, a primer, with a 3' end for DNA polymerase to perform replication. DNA polymerase can only add new nucleotides to an existing strand of DNA (primer).
RNA can be made without a primer.
DNA Polymerase only works for the 3' end. This is where the 5' to 3' synthesis name orginated.
Mechanism of replication
DNA replication reaction mechanism by Allen Gathman, on Flickr
See video explaining this
Elongation of the DNA chain is catalyzed by the DNA polymerases, specifically polymerase III. This is driven by the nucleophilic attack of the primer's 3' hydroxyl group on the 5' dNTP's alpha phosphate. In synthesizing DNA from 5' to 3', a leaving group is provided(consisting of the magnesium ion and pair of phosphates) causing a shift in distribution of the bond on the dNTP's alpha phosphate. This makes it easier to remove the pair of phosphates through hydrolysis, thus releasing energy to catalyze the reaction to create the phosphodiester bonds in DNA synthesis.
Pyrophosphate is a high energy compound with a low concentration in the cell, because it is easily broken down into two inorganic phosphates when reacted with water. This allows the reaction to move in the forward direction.
Magnesium removes the bond from the phosphate by altering the electron distribution and creating a weaker polar bond. This allows the OH group from 3' C to perform nucleophilic attack on phosphate and thus forming a phosphodiester double bond.
Why not at the 5' end?
3' -> 5' Replication won't work by Allen Gathman, on Flickr
3' to 5' does not work because for the nucleophilic attack to occur, there has to be a good leaving group. When added to the 5' end, the additional phosphate groups serve as good leaving groups, if the second nucleotide were added to the 3' end, however, then nucleophilic attack cannot occur because the oxygens are tightly attached to the Phosphorous. Therefore the addition of the nucelotides is always done from the 5'-3'.
Prokaryotic replication fork by Allen Gathman, on Flickr
The replication fork has a leading strand and also a lagging strand.
The lagging strand requires new primers to be made in order to synthesize DNA gaps of DNA fragments. The enzyme DNA Polymerase I is responsible for removing the nicks and gaps between fragments. In this case, a "gap" refers to missing nucleotides in the new strand, while a "nick" denotes a missing phosphodiester bond between two nucleotides.
The lagging strands between the "gaps" and "nicks" are called Okazaki Fragments. These lagging strands are replicated in pieces due to its moving in the opposite direction of the replication fork. As we know a strand can not be replicated from 3' to 5', so these RNA primers form at the 5' starting point. Besides converting the nicks between the Okazaki Fragments into gaps, DNA Polymerase I has another job. It removes the RNA primer from the 5' end of the lagging strand. Once Pol I has completely removed the RNA primer and converted the gap into a nick, ligase uses ATP to catalyze the formation of a phosphodiester bond linking the Okazaki fragments.
Within the picture giving a basic idea of a replisome, dnaB is the helicase which separates the strands of DNA; primase is dnaG which makes RNA primers, and dnaC is also influential in the process. This group of proteins functioning together makes up a preprimosome. The Polymerase III then is able to add on nucleotides to the RNA primers with the support of the sliding clamp which is made of 6 proteins and holds Polymerase III in place.
This process is further explained at DNA replication in bacteria