DNA replication
DNA replication has been outlined in section 3.4.1 . which should be reviewed before tackling this section. This HL section provides more detail on the process of DNA replication which takes places during the S section of the Interphase.
The models of DNA replication are based on some prokaryotic organisms such as E.coli. The diversity of this group however would suggest that we should be cautious in extrapolating the mechanism to the whole group. Eukaryotic organisms have more complex mechanism although they share the same broad mechanism.
Students should pay close attention to the orientation of the nucleotides when the DNA chain is polymerised.
7.2.1 Direction of DNA replication.
7.2.2 Prokaryotic DNA replication.
7.2.3 Eukaryotic DNA replication
7.2.1 State that DNA replication occurs in a
5’→ 3’ direction.(1)
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DNA replication has been outlined in section 3.4.1 .
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The 5' (prime) end of the free nucleotide is added to the 3' (prime) end of the nucleotide chain that is already formed.
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(a) Shows a nucleotides in isolation. The 5 ' is orientated to the 3' so that DNA polymerase enzyme can form the phosphodiester bond between carbon 5 and carbon 3.
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(b) There is a polynucleotide chain already in place. Note the position of the free 3' on the left end of this chain.
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The free nucleotide 5' end is bonded covalently to the 3' end on the already formed polynucleotide chain.
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7.2.2 Explain the process of DNA replication
in prokaryotes, including the role of
enzymes (helicase, DNA polymerase,
RNA primase and DNA ligase), Okazaki
fragments and deoxynucleoside
triphosphates.(3)
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The diagram shows the loop DNA of a prokaryotic organism.
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Ori is the point of origin (start) for DNA replication.
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Ter is the point at which the replication will finish.
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The DNA replication takes place under the control of a number of different proteins and enzymes here indicated as replication complex.
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In E. coli five such polymerases have been identified with DNA polymerase III being associated with most polymerisation of the pentose-phosphate backbone. Humans have as many as fourteen polymerases.
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Until recently it was thought that the replication complex moved over the DNA. New evidence now suggests that the replication complex is static and that the DNA is moved.
(c) DNA molecule
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Shows the two anti-parallel polynucleotide chains.
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Note the position of the 5' and 3' ends of the chains but remember when working out the direction of replication we focus on the NEW strand.
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The parent strands of polynucleotide are coded red and blue in this diagram for clarity.
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New strands will be coloured green for ease of identification.
(d) DNA Helicase enzyme
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The enzyme is unwinding the chain and breaking the bonds between the complementary base pairs (A-T, G-C).
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The position of the helicase and the opening of the DNA helix is called a replication fork.
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The helicase has parted the two polynucleotide chains.
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(e) This red original 'parent' strand is polymerised at the same time as the one below but for the purposes of this diagram is left till later in our model.
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The diagrams follows the building of the new polynucleotide chain on the blue side first. 3' prime to 5' prime on the original DNA strand.
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A free nucleotide (Green 1) complementary base pairs with the first 'blue' nucleotide in the parent strand.
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The free nucleotide (Green 2) complementary base pairs.
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DNA polymerase III joins the 5' to the 3' of the NEW STRAND.
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Prokaryotic DNA polymerases can work at around 1000 bases per second which means the whole circular (loop) can replicated between 20 and 40 minutes.
.gif)
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(g) The nucleotide sequence is building up as a new polynucleotide using the original as a template.
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Specificity is maintained through complementary base pairing of A-T, G-C.
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The next nucleotide (Green 3) has base paired and is being polymerised by DNA polymerase III.
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The helicase is progressing just ahead of the DNA polymerase III creating the replication fork.
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Errors do occur in the replication process but there are biochemical proof reading, repairing and removal mechanisms.
Retuning to the other parent polynucleotide (red).
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Since this was anti-parallel to the blue strand the template nucleotides (red) have the opposite direction to the blue ones.
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(h) Free nucleotides have complementary base paired with the first two template bases.
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Notice that the nucleotides cannot be joined as DNA polymerase is specific to joining 5' to 3'.
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DNA polymerase III like all enzymes has an active site that is specific to the 5' to 3' orientation of the two nucleotides to be joined.
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The chain is not polymerised at this stage.
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The bases continue to add working in behind the DNA helicase enzyme. (Upper Green 1-4).
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Below we have the polymerisation of the new strand on the 'blue' template as previously described in part (g).
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(L) The red strand in which the nucleotides do not immediately polymerise is called the Lagging strand (since it lags behind) it forms on the "5 to 3' original DNA template.
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(m) Note that there are now a number of bases already in position forming the lagging strand (in reality this is about 100-200 nucleotides).
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DNA polymerase III can now work 'backwards' towards the ori joining the sugar phosphate backbone of the polynucleotide.
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On the lagging strand the DNA polymerase is working away from the replication fork.
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(n) On the Leading strand (new strand building on the blue template) the DNA polymerase III works towards or follows the helicase.
The important point to note here is that the DNA polymerase only works by joining 5' nucleotides to 3' nucleotides on the established chain.
The lagging strand is therefore made up of a number of short polynucleotide chains that need joining together.
The short chains are called Okazaki fragments after the Japanese Biochemist Reiji Okazaki.
Summary:
This diagram is the usual version used to describe the process of leading and lagging strand polymerase activity.
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( p) shows the orientation of the DNA helix (with helicase) for the diagram below.
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(q) Note the position of the replication fork with the DNA helicase opening the DNA chain.
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(r) The leading strand forming with DNA polymerase III
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On the lagging strand (top green) the new strand is presented as a number of Okazaki fragments.
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(s) The DNA polymerase III on this strand has to work from the beginning of each fragment towards the 3' free end of the lagging strand (away from the replication fork).
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DNA ligase is the enzyme that joins the fragments.
Remember always think about the action of the DNA polymerase III adding the 5' of the free nucleotide to the 3' of the already established new strand. This single fact allows the process to be tracked and alternative diagrams to be interpreted.
Primers:
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In fact all polymerisation both leading and lagging strands actually begin with the addition of 'priming' RNA nucleotides.
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(u) RNA nucleotides (yellow) attach to the first few bases on the template through the action of a Primase enzyme.
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DNA polymerase III then adds DNA nucleotides to the Primer (v).
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Later the RNA primer is broken down and removed by DNA polymerase I
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DNA nucleotides are added to replaced the removed RNA nucleotides.
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The Pentose -phosphate backbone is joined by DNA ligase.
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7.2.3 State that DNA replication is initiated
at many points in eukaryotic
chromosomes.(1)
Prokaryotic DNA polymerase can work at around 1000 bases per second which means the whole circular (loop) can replicated between 20 and 40 minutes.
The eukaryotic DNA polymerase works much slower around 50 bases per second. With as many as 80 million bases to replicate the job is achieved in about one hour by having many replication forks
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