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DNA polymerase and the replication fork

What is replication?

 

DNA replication is the production of two DNA double helices that are identical to each other but made from one original helix. The method of replication is semi-conservative; this means that in the two daughter helices one strand of each is comprised of parental DNA (from the original helix), and the other is comprised of newly synthesised DNA. Synthesis occurs at the replication fork, our focus in this article. Replication is a complex process, involving many, many different enzymes - such as DNA polymerase - which have frustratingly similar names to enzymes in other processes.

 

Here is a short video which is really useful for understanding semi-conservative replication, especially if you're confused about which strands are parental or newly synthesised: http://www.youtube.com/watch?v=WrWs4IIezGA&feature=relmfu

 

*All enzyme names in this article will be in bold so they're easy to pick out.

** To enlarge the images please click on them and zoom in.

Replication in humans (eukaryotes)

SSBs, primers, polymerases

Before replication at the fork can begin, the helix needs to be unwound, so that it is straight like a ladder. As DNA is supercoiled (or plectonemic), the whole chromosome would need to be completely unwound before any of it would be straight. This would not be very practical. Eukaryotes avoid this by utilising an enzyme called topoisomerase I. This enzyme makes a single-stranded cut through the DNA, allowing a small section of DNA to be opened up and straightened out (figure 1). This straight section will become the replication fork, which is found at the origin of replication and is where topoisomerase works.

 

Replication always progresses from the 5' end of the DNA backbone to the 3' end, with new nucleotides being added onto the 3' site. To start this nucleotide addition, an enzyme named helicase must break the hydrogen bonds between the complementary bases on each of the parent strands, in the gap that topoisomerase created (figure 2). This whole process is much easier to understand if you now think of the opened up DNA as a capital Y, with the tail of the Y being the DNA that hasn't been separated by helicase yet. Think of each arm of the Y then as a single parent strand, running anti-parallel to one another. This is where new DNA will be synthesised. Synthesis on one of the arms will move toward the replication fork as this arm runs 3'->5' and complementary replication will move in the opposite direction (5'->3') - away from the replication fork. The former is known as the leading strand and encounters far fewer problems than the latter which is known as the lagging strand (figure 3).

 

To prevent the two arms of the Y from joining back together again due to the attraction between them, single stranded binding proteins (SSBs) bind to each strand (figure 3). Events on the leading strand are simple; an enzyme known as primase will start off replication by adding RNA nucleotides from 5'->3' on the new DNA strand until around 15 nucleotides have been added. This short section of RNA is known as a primer. Here, another enzyme called DNA polymerase alpha will extend the primer by around 20 more DNA nucleotides. After around a total of 35 bases have been added, the main replicating enzyme in eukaryotes - called DNA dependent DNA polymerase delta - will take over. This enzyme is DNA dependent as it needs existing DNA as a template (parent strand) to polymerise new DNA. DNA polymerase delta can only attach to the DNA if a primer is present, creating a double stranded section for it. It is held on tightly to the DNA by a complex called the Proliferating Cell Nuclear Antigen (PCNA), which also helps it to attach and detach on the lagging strand. Now all that's left to complete replication on this strand is for DNA polymerase to move 5'->3' adding nucleotides as it goes until it reaches the end of the chromosome. This is one continuous process, unlike on the lagging strand (figure 4).

 

On the lagging strand, the same enzymes are used to create new DNA. The difference is that it has to be carried out in fragments - known as Okazaki fragments - which are joined up later. This happens as the parental DNA strand runs 3'->5' in the same direction as the replication fork moves, which means complementary 5'->3' replication will move against the fork. Replication will constantly have to jump backwards as the fork progresses. DNA polymerase delta would keep jumping backward extending primer after primer until the whole strand had been replicated. The only problem with this is that none of the fragments would be joined and there would be primers everywhere, so instead DNA polymerase removes them and joins fragments as it goes (figure 5).

 

On any fragment with one in front DNA polymerase delta will synthesise DNA until it bumps into the primer ahead. Here helicase will break base pairs again up until the DNA ahead of the primer that DNA polymerase alpha originally synthesised (figures 6-7). DNA polymerase delta will then continue adding bases until it reaches the one base where the primer is still being held on (known as the branch point). Here FEN1 (an endonuclease) will chop at that base, cutting the primer and a small section of DNA off; this leaves a one base pair gap. FEN1 can't cut through the RNA primer, only DNA, and that's why it must chop off that little bit of DNA too (figure 7). To finish off, an enzyme named DNA ligase will close up this gap, joining the two Okazaki fragments (figure 8). The PCNA will then move DNA polymerase delta backward to start this process again with the next fragment. This is repeated until the whole strand is completed.

 

It should be noted that a problem arises at the end of the chromosome, on the lagging strand. The last few bases at the end of the strand can't be synthesised as the primer has nowhere to attach to, the parental DNA doesn't extend far enough to make a primer (figure 9). To fix this problem an enzyme called telomerase extends the overhang on the parental strand far enough so that a primer can be added just before the bases that need to be synthesised. This allows the last fragment to be made and ensures that the chromosomes don't get any shorter during replication. Telomerase adds repeating units of TTAGGG in humans, and this sequence is different in every species (figure 10).

 

Here is a video which will really help to visualise the process if the whole 5', 3' thing is confusing. It has a few extra points than in this article and it uses different names for some of the enzymes, but the process is still the same: http://www.youtube.com/watch?v=i2kEHnBOA-Q

 

FEN1, ligase, telomerase

Differences between eukaryotes and E.coli (a prokayote)

Humans

  • Topoisomerase I makes a single stranded cut in the DNA;
  • Primase and DNA polymerase alpha make the primer;
  • DNA polymerase delta is the main replicating enzyme;
  • FEN1 and ligase join up the two fragments;
  • PCNA moves the main replicating enzyme along the lagging strand.

 

FEN1 is an endonuclease, it chops right through the DNA (after the primer) hence the name endo: -meaning inside.

    E.coli

    • Topoisomerase II makes a double stranded cut in the DNA;
    • Only primase makes the primer;
    • DNA polymerase III is the main replicating enzyme;
    • DNA polymerase I and ligase join up the two fragments;
    • The gamma complex moves the main replicating enzyme along the lagging strand.

     

    DNA polymerase I is an exonuclease; when removing the primer instead of creating a branch point and chopping through the DNA, it steps in front of DNA polymerase III and snips off the primer bases (from 5' to 3'). It does this one at a time with DNA polymerase III following it, adding bases one by one. Then, like in humans, the last base of each fragment is joined up by ligase.

      References

      These references for the article also happen to be extremely useful textbooks, so get hold of one if you can!

      Alberts, B; et al. (2010). Essential cell biology, 3rd ed. Garland Science, New York and London.

      Brown, T (2011). Introduction to genetics: A molecular approach. Garland Science, New York and London.

      Hartl, D. (2011). Essential genetics: A genomics perspective, 5th ed. Jones and Bartlett publishers, Sudbury, Massachusetts.

      Krebs, J; Goldstein, E; Kilpatrick, S. (2011). Lewin's genes X, 10th ed. Jones and Bartlett publishers, Sudbury, Massachusetts.

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