Chapter 9: DNA Replication
9.1 DNA Replication is Semiconservative
Chapter 9 deals with the process of DNA Replication. In this first section, we will discuss the
semiconservative nature of DNA replication
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This diagram gives a nice overview of the major enzymes and proteins involved with DNA replication. Once the Ori has been opened, exposing single stranded DNA a ...
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Chapter 9: DNA Replication
9.1 DNA Replication is Semiconservative
Chapter 9 deals with the process of DNA Replication. In this first section, we will discuss the semiconservative nature of DNA replication
DNA Replication is Semiconservative Before the nature of DNA replication was known, there were three putative hypotheses that could describe the nature of the DNA replication process. In the fist hypothesis, the daughter strand is created in a completely new manner while retaining the structure of the original parent strands. This is a conservative replication model. The second model is a semiconservative model, where the resulting products each contain one parent strand of DNA and one new strand of DNA. The third model for DNA replication is dispersive, where the parent DNA would be interspersed with newly replicated sequences of DNA.
Chapter 9: DNA Replication
9.2 DNA Replication in Prokaryotes And with that introduction to one of the earliest studies about DNA replication, we will now dive into some of the key details of prokaryotic replication.
Prokaryotic DNA is circular One of the major features of prokaryotic DNA is the it is circular in nature, and the size of the chromosomal DNA is much smaller than eukaryotic counterparts. Thus, replication is typically characterized by having one origin of replication, called Ori, in the diagram. The ori is the site where the DNA double helix opens up and each of the strands is used as a template to synthesize the new daughter strands (shown as the dashed circles in the diagram on the right. Notice the replication is bidirectional and moves outward from the Ori in both directions..
Prokaryotic Replication Overview This diagram gives a nice overview of the major enzymes and proteins involved with DNA replication. Once the Ori has been opened, exposing single stranded DNA a DNA replication fork can form. Leading this way is the DNA helicase enzyme that is involved in unwinding the double stranded DNA to create the single stranded bubble. When the helicase unwinds the DNA, this introduces more tension into the double stranded DNA ahead of the replication fork. The DNA topoisomerase enzymes are used to relieve this supercoiled tension during the replication process. ssDNA binding proteins will bind to the DNA and keep it from reforming the double helix. Note that each strand of DNA is lying in the opposite orientation, one in the 5โ to 3โ direction and the other in the 3โ to 5โ direction. Recall that DNA synthesis only occurs in the 5โ to 3โ direction with the insertion of the new nucleotide at the 3โ-OH of the previous base. Thus, for bidirectional synthesis to occur one strand can move easily in the same direction that the replication fork is going. This is the leading strand. This is where the new strand can be built in the 5โ to 3โ direction. The lagging strand has to be built in short bursts in the opposite direction of the replication fork as the lagging strand template must wind itself backwards through the DNA polymerase enzyme to link in bases in the 5โ to 3โ direction. After a short region is completed, the DNA is released and rewound again upstream. This creates multiple smaller DNA fragments on the lagging strand, known as Okazaki fragments, during the replication process. The DNA polymerase enzyme requires a few key components to mediate DNA replication. It must have a template strand to know which base to incorporate into the growing strand, and it
must also have a primer to be able to incorporate the next base onto. DNA replication cannot just happen on its own. Thus, small RNA segments will serve as a primer that will enable the start of DNA replication. The RNA primase enzyme mediates this function. Only one primer is needed for the leading strand to start, but multiple small primers will be needed on the lagging strand, each time the sequence is released. Another DNA polymerase enzyme will end up removing the RNA primer sequences and filling them in with the appropriate DNA and the sugar/phosphate backbone will be resealed by the DNA ligase enzyme. Within the next few slides we will take a look at this process in more detail.
Primosome This diagram shows the assembly of the DNA primosome. This involves the binding of an initiator protein to the Ori sequence. In E. coli, this initiator is encoded for by the DnaA gene. The DnaA initiator protein binds to ATP and specific sequences at the Ori. DnaA is a long snake-like protein with multiple subunits. Domains III and IV are the most visible. Binding of DnaA + ATP causes torsional strain at the Ori and initiates the unwinding process. DnaC (the helicase loader) Interacts with DnaA that is bound at the Ori, and also traps the DnaB helicase protein in an open lock washer conformation so that it can load onto the ssDNA. Domain I of DnaA helps to capture another DnaB-DnaC complex to load on the opposite strand. Once this is assembled, DnaC (the helicase loader) dissociates from the complex and DnaB shifts conformation to a closed ring structure. This forms the functional primosome. The DnaB helicase then begins to unwind the DNA at the new replication fork, and the DnaG primase enzyme adds an RNA primer.
DNA Polymerase Enzymes E. coli has a total of 5 DNA Polymerase enzymes, 3 of which are involved in DNA replication (I, II, and III). DNA polymerase III is the main polymerase involved in both leading strand biosynthesis and the synthesis of the Okazaki Fragments during DNA replication. The DNA polymerase III holoenzyme is comprised of 10 different proteins organized into three functionally distinct, but physically interconnected assemblies: (1) the ฮฑฮตฮธ core, (2) the ฮฒ 2 sliding clamp, and (3) the ฮดฯnฮณ3-nฮดโฮจX clamp loader complex. Figure a, shows the standard textbook model of a DNA Replisome with the coupled and highly coordinated processes of leading strand and lagging strand synthesis. DNA polymerase III is connected to the DnaB helicase through the ฯ subunit of the clamp-loader comples and two or three polymerase cores replicate DNA from both leading strand and lagging strand DNA templates concurrently. The ssDNA in the lagging strand loop is bound by ssDNA binding proteins (SSB). However, as shown in Figure b, recent studies have shown that E. coli DNA polymerase III is readily exchangeable at the fork and that leading strand and lagging strand synthesis may not be tightly coupled, or may even be accomplished by different DNA polymerase III holoenzymes. The DnaB helicase can also be decoupled from the DNA polymerase complex and translocate ahead of the apex of the fork.
DNA Pol III
- Polymerase
- Proofreading DNA Pol III polymerase activity is shown here with the incorporation of the deoxythymidine triphosphate into the growing chain.
DNA Pol III
- Polymerase
- Proofreading Occasionally there is an extension error, where the wrong nucleotide is incorporated into the growing chain. Here you can see that an โAโ has been incorporated opposite a โCโ, rather than the correct โGโ base.
DNA Pol III Core Cryo-EM structures of the DNA Pol III Core, clamp and the tao clamp loader are shown here, with the DNA polymerase in both the polymerization and proofreading modes. This shows that there is a lot of flexibility in the DNA polymerase, especially in the thumb region, such that the polymerase is essentially adding a base, and then shifting to check the added base, before it moves forward to add the next base.
DNA Polymerase III This diagram is showing a side view of the DNA polymerase III protein, with special detail shown for the flexible movement of the tao binding domain (TBD) and the oligonucleotide binding domain (OB) when the polymerase is free or with the DNA bound to the structure.
DNA Polymerase I DNA Polymerase I is mainly involved with primer removal and replacement during DNA replication, as well as with DNA Proofreading. In addition to the 5โ to 3โ polymerase active and the 3โ to 5โ exonuclease proofreading abilities, the DNA pol I enzyme also has 5โ to 3โ exonuclease activity. This means that the DNA Polymerase I can remove the RNA primer sequences that are laid down along the lagging strand during DNA replication, and replace that sequence with DNA. The E. coli DNA Pol I is shown on the left and was crystalized without the 5โ to 3โ exonuclease domain. The green outline shows where this portion of the protein normally would be. The structure of the right is the full DNA pol I enzyme from Thermus aquaticus. Note that DNA Pol II is predominantly utilized in the repair of mismatch bases following DNA replication, and has some overlapping function with DNA Pol I.
DNA Polymerase I Figure from: Biology. Libretexts Once the DNA Polymerase III has finished synthesizing the bulk of the DNA, you will notice that the lagging strand of each replication fork, has many RNA primers throughout the DNA. These cannot be removed by DNA polymerase III, as it does not have 5โ โ 3โ exonuclease activity. Thus, when DNA polymerase III runs into the downstream primer, it falls off of the DNA and is finished with its synthesis ability. The DNA Polymerase I will link on to the DNA and scan along the sequence checking the 5โ to 3โ strands. It has 5โ to 3โ exonuclease activity and can remove and replace the RNA primers in the Okazaki fragments. The one thing that it cannot do, it cannot seal the breaks in the backbone between the separate Okazaki fragments. The DNA Ligase enzyme will be required for this purpose, and weโll take a look at that over the next few slides.