DNA Replication

Meslelson-Stahl Experiment

Three hypotheses had been previously proposed for the method of replication of DNA.

In thesemiconservativehypothesis, proposed byWatsonandCrick, the two strands of a DNA molecule separate during replication. Each strand then acts as a template for synthesis of a new strand.

Theconservativehypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one. According to this model,histoneproteins bind to the DNA, revolving the strand and exposing the nucleotide bases (which normally line the interior) for hydrogen bonding.

Thedispersivehypothesis is exemplified by a model proposed byMax Delbrück, which attempts to solve the problem of unwinding the two strands of the double helix by a mechanism that breaks the DNA backbone every 10 nucleotides or so, untwists the molecule, and attaches the old strand to the end of the newly synthesized one. This would synthesize the DNA in short pieces alternating from one strand to the other.

Each of these three models makes a different prediction about the distribution of the "old" DNA in molecules formed after replication. In the conservative hypothesis, after replication, one molecule is the entirely conserved "old" molecule, and the other is all newly synthesized DNA. The semiconservative hypothesis predicts that each molecule after replication will contain one old and one new strand. The dispersive model predicts that each strand of each new molecule will contain a mixture of old and new DNA.

The Meselson-Stahl Experiment

TheMeselson–Stahl experimentwas an experiment byMatthew MeselsonandFranklin Stahlin 1958 which supported the hypothesis thatDNA replicationwassemiconservative. In semiconservative replication, when the double stranded DNA helix is replicated each of the two new double-strandedDNAhelixes consisted of one strand from the original helix and one newly synthesized. It has been called "the most beautiful experiment in biology.

Nitrogenis a major constituent of DNA.14Nis by far the most abundantisotopeof nitrogen, but DNA with the heavier (but non-radioactive)15Nisotope is also functional.

E. coliwere grown for several generations in a medium with15N. When DNA is extracted from these cells and centrifuged on a salt density gradient, the DNA separates out at the point at which its density equals that of the salt solution. The DNA of the cells grown in15N medium had a higher density than cells grown in normal14N medium. After that,E. colicells with only15N in their DNA were transferred to a14N medium and were allowed to divide; the progress of cell division was monitored by microscopic cell counts and by colony assay.

DNA was extracted periodically and was compared to pure14N DNA and15N DNA. After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of15N DNA, and one of14N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of15N and14N DNA, either of which would have appeared as DNA of an intermediate density.

The authors continued to sample cells as replication continued. DNA from cells after two replications had been completed was found to consist of equal amounts of DNA with two different densities, one corresponding to the intermediate density of DNA of cells grown for only one division in14N medium, the other corresponding to DNA from cells grown exclusively in14N medium. This was inconsistent with dispersive replication, which would have resulted in a single density, lower than the intermediate density of the one-generation cells, but still higher than cells grown only in14N DNA medium, as the original15N DNA would have been split evenly among all DNA strands. The result was consistent with the semiconservative replication hypothesis.

  • Generation 0 is grown in the heavy (15N) Nitrogen. This is represented by the blue strands.
  • It is then moved to a mixture of light (14N) Nitrogen – red strand. Now when the DNA replicates the new strand will be contain the 14N. This means that the old strand contains 15N and the new strand contains 14N.
  • The next two generations are both kept in light (14N) nitrogen and so as more DNA replicates, the new strands are all made of 14N – the red strands.

DNA Replication Mechanism in Prokaryotes

Watson and Crick first reasoned that complementary base pairing provides the basis of fidelity inDNAreplication; that is, that each base in thetemplatestrand dictates the complementary base in the new strand. However, we now know that the process of DNA replication is very complex and requires the participation of many different components. Let’s examine each of these components and see how they fit together to produce our current picture of DNA synthesis inE. coli,the best-studied cellular replication system. In the preceding section, we introduced the concept of thereplication fork.Figure 1gives a detailed schematic view of fork movement during DNA replication; we can refer to this illustration as we consider each component of the process.

DNA Polymerases:

In the late 1950s, Arthur Kornberg successfully identified and purified the firstDNA polymerase, anenzymethat catalyzes thereplicationreaction.

This reaction works only with the triphosphate forms of the nucleotides (such as deoxyadenosine triphosphate, or dATP). The total amount ofDNAat the end of the reaction can be as much as 20 times the amount of original input DNA, so most of the DNA present at the end must be progeny DNA.Figure 2depicts the chain-elongation reaction, orpolymerizationreaction, catalyzed by DNA polymerases. We now know that there are three DNA polymerases inE. coli.The firstenzymethat Kornberg purified is calledDNA polymeraseIorpol I.This enzyme has three activities, which appear to be located in different parts of the molecule:

1. a polymerase activity, which catalyzes chain growth in the 5′ → 3′ direction;

2. a 3′ → 5′exonucleaseactivity, which removes mismatched bases; and

3. A5′ → 3′exonucleaseactivity, which degrades double-strandedDNA.

Subsequently, two additional polymerases, pol II and pol III, were identified inE. coli.Pol II may repair damagedDNA, although no particular role has been assigned to thisenzyme. Pol III, together with pol I, has a role in thereplicationofE. coliDNA. The complete complex, orholoenzyme,of pol III contains at least 20 differentpolypeptidesubunits, although the catalytic “core” consists of only three subunits, alpha (α), epsilon (ϵ), and theta (θ).

DNA polymerase III holoenzymeis the primaryenzymecomplex involved inprokaryoticDNA replication. It was discovered byThomas Kornberg(son ofArthur Kornberg) andMalcolm Gefterin 1970. The complex has high processivity (i.e. the number ofnucleotidesadded per binding event) and, specifically referring to the replication of theE.coligenome, works in conjunction with four other DNA polymerases (Pol I,Pol II,Pol IV, andPol V). Being the primaryholoenzymeinvolved in replication activity, the DNA Pol IIIholoenzymealso has proofreading capabilities that correct replication mistakes by means ofexonucleaseactivity working 3'→5'. DNA Pol III is a component of thereplisome, which is located at the replication fork.

The replisome is composed of the following:

  • 2DNA Pol III enzymes, each comprisingα,εandθsubunits. (It has been proven that there is a third copy of Pol III at the replisome.[1])
  • the α subunit (encoded by thednaEgene) has the polymerase activity.
  • the ε subunit (dnaQ) has 3'→5' exonuclease activity.
  • the θ subunit (holE) stimulates the ε subunit's proofreading.
  • 2βunits (dnaN) which act as slidingDNA clamps, they keep the polymerase bound to the DNA.
  • 2τunits (dnaX) which acts to dimerize two of the core enzymes (α, ε, and θ subunits).
  • 1γunit (also dnaX) which acts as a clamp loader for the lagging strandOkazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit (holA), and 1 δ' subunit (holB). The δ is involved in copying of the lagging strand.

Χ(holC) andΨ(holD) which form a 1:1 complex and bind to γ or τ.

Activity

DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second.[3]DNA Pol III activity begins after strand separation at the origin of replication. Because DNA synthesis cannot startde novo, anRNA primer, complementary to part of the single-stranded DNA, is synthesized byprimase(anRNA polymerase)

Prokaryotic Origin of Replication

E. colireplicationbegins from a fixedoriginbut then proceedsbidirectionally(with moving forks at both ends of the replicating piece), as shown inFigure 3, ending at a site called theterminus.The unique origin is termedoriC is 245 bp long and has several components, as illustrated inFigure 8-23. First, there is a side-by-side, or tandem, set of 13-bp sequences, which are nearly identical. There is also a set of binding sites for a protein, the DnaA protein. An initial step inDNAsynthesis is the unwinding of the DNA at the origin in response to binding of the DnaA protein. The consequences of bidirectional replication can be seen inFigure 8-24, which gives a larger view of DNA replication.

Priming DNA synthesis

DNApolymerases can extend a chain but cannot start a chain. Therefore, as already mentioned, DNA synthesis must first be initiated with aprimer, or shortoligonucleotide, that generates a segment of duplex DNA. The primer in DNAreplicationcan be seen inFigure 3(see alsoFigure 8-20).RNAprimers are synthesized either byRNA polymeraseor by anenzymetermedprimase. Primase synthesizes a short (approximately 30 bp long) stretch of RNA complementary to a specific region of thechromosome. The RNA chain is then extended with DNA byDNA polymerase.E. coliprimaseforms a complex with thetemplateDNA, and additional proteins, such as DnaB, DnaT, PriA, Pri B, and PriC. The entire complex is termed aprimosome.

Leading strand and Lagging Strand

DNApolymerases synthesize new chains only in the 5′ → 3′ direction and therefore, because of theantiparallelnature of the DNA molecule, move in a 3′ → 5′ directionon thetemplatestrand.The consequence of this polarity is that while one new strand, theleading strand, is synthesized continuously, the other, thelagging strand, must be synthesized in short, discontinuous segments, as can be seen inFigure 4. The addition of nucleotides along the template for thelagging strandmust proceed toward the template’s 5′ end (becausereplicationalwaysmoves along the template in a 3′ → 5′ direction so that the new strand can grow 5′ → 3′). Thus, the new strand must grow in a direction opposite that of the movement of thereplication fork. As fork movement exposes a new section of lagging-strand template, a new lagging-strand fragment is begun and proceeds away from the fork until it is stopped by the preceding fragment. InE. coli,pol III carries out most of the DNA synthesis on both strands, and pol I fills in the gaps left in the lagging strand, which are then sealed by theenzymeDNAligase.DNA ligases join broken pieces of DNA by catalyzing the formation of aphosphodiester bondbetween the 5′ phosphate end of a hydrogen-bondednucleotideand an adjacent 3′ OH group. It is the only enzyme that can seal DNA chains.Figure 5 shows the lagging-strand synthesis and gap repair in detail. The primers for the discontinuous synthesis on the lagging strand are synthesized byprimase(step a). The primers are extended byDNA polymerase(step b) to yield DNA fragments that were first detected by Reiji Okazaki and are termedOkazaki fragments.The 5′ → 3′exonucleaseactivity of pol I removes the primers (step c) and fills in the gaps with DNA, which are sealed by DNA ligase (step d). One proposed mechanism that allows the same dimeric holoenzyme molecule to participate in both leading- and lagging-strand synthesis is shown inFigure 8-31. Here, the looping of the template for the lagging strand allows a single pol III dimer to generate both daughter strands. After approximately 1000 base pairs, pol III will release the segment of lagging-strand duplex and allow a new loop to be formed.

Helicases and Topoisomerases

Helicasesare enzymes that disrupt the hydrogen bonds that hold the twoDNAstrands together in adouble helix. Hydrolysis ofATPdrives the reaction. AmongE. colihelicases are the DnaB protein and the Rep protein. The Rep protein may help to unwind the double helix ahead of the polymerase (refer toFigure 8-20). The unwound DNA is stabilized by theexplain whyreplicationis in the 5′ → 3′ direction. As we saw earlier, new bases are added when the 3′ OH on the terminal deoxyribose of the growing strand attacks the high-energy phosphate of thenucleotidetriphosphate that is being added (seeFigure 8-21). Chain growth is thus 5′ → 3′. It is conceivable that replication could be in the 3′ → 5′ direction, the 5′ triphosphate at the bottom would be the last base on the chain, and the 3′ OH that attacks it would be on the free nucleotide triphosphate about to be added to the strand). However, if replication were in this direction, there would be exonuclease excisions at the 5′ end of the strand. When a mismatched base was removed, a 5′ OH would be left at the end of the growing strand. The 3′ OH of an incoming nucleotide triphosphate would thus be facing this 5′ OH instead of the high-energy 5′ triphosphate necessary for bond formation. No bond would form and strand growth would stop. Therefore, replication is not in the 3′ → 5′ direction.

In E. coli, Tus (Terminus utilization substance) is a protein that binds to terminator sequences. Tus binds to 10 closely related sites encoded in the chromosome know as Ter sites. The bound Tus protein effectively haltsDNA polymerasemovement. Ter sites are designated TerA, TerB, TerC…… TerJ. Finally topoisomerase IV unlinks the two circular DNA duplexes.The multipleTersites in the chromosome are oriented such that the two oppositely moving replication forks are both stalled in the desired termination region.