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Module: BIOM - 1006

Lecturer: Dr Sherwood

Date: 21/11/16

DNA Replication
o

DNA replication occurs during cell division
...
First, they
decided to differentiate between hybrid and non-hybrid helices
 They grew two batches of E
...
This is because each base only pairs to their
counterpart
 The new strand created is complementary to the template strand

o

One of the main enzymes involved in this replication is DNA polymerase
 This is involved in the polymerisation of deoxyribonucleotide triphosphates (dNTPs) (see below)
 The N can be adenine, cytosine, guanine or thymine
 The polymerase can only add dNTPs to the 3’ end of the new growing DNA strand
 Hence, the synthesis is 5’ to 3’
 The two furthermost phosphates are removed before the nucleotide is added to the 3’ end of the
strand via the catalysis of the covalent linkage between the two nucleotides
 A pyrophosphate is released

o

The DNA polymerase makes an error roughly every 107 nucleotides
 Fortunately, the polymerase recognises this, changes shape and allows another active site on the
enzyme to excise the faulty addition
 Every nucleotide is checked
 A mistake that goes unrecognised will produce a mutation

o

To begin the synthesis of a new strand of DNA, an
initiator protein must bind to the replicator origins
 These replicator regions are specific sequences
of DNA
 The initiator proteins pull the two strands
apart
 Less energy is required to pull a few
base pairs apart than to pull the entire
helix apart
 This produces two replication forks (see right)
 The forks move in opposite directions

o

This produces a problem however, if the strands run antiparallel and the polymerase only works in the 5’ to
3’ direction
 The leading strand that runs 5’ to 3’ is synthesised continuously
 The lagging strand is synthesised discontinuously

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Bits are synthesised and then stitched together

o

The DNA polymerase produces loops in the DNA called Okazaki fragments
 This allows the polymerase to run for short stretches in a 5’ to 3’ direction
 The process of synthesising the DNA on the lagging strand is as follows
 An RNA primer (~10 nucleotides) is added to the lagging strand
 This is a short strand of complementary RNA created by the enzyme primase (this is
a subcategory of enzymes known as RNA polymerase)
 The polymerase then creates short segments of dsDNA from the Okazaki fragments
 A new primer is needed for every fragment (every ~200 nucleotides)
 The RNA segment must now be removed and replaced with DNA
 The RNA is removed from the strand using a nuclease enzyme
 DNA then replaces it using the enzyme ‘repair nuclease’ (this uses the end of
the former Okazaki fragment as a primer)
 Finally, DNA ligase will join the 5’ phosphate of one DNA fragment to the 3’
hydroxyl of the other
o The RNA primer often contains many mistakes because the polymerase
does not proof read it, it is removed anyway so it does not cause much of a
threat

o

Many other proteins are also used in DNA replication:
 DNA topoisomerase is used to produce transient nicks in the phosphate backbone of the DNA to
be split open by helicase
 This reduces the tension on the DNA and causes less damage
 A sliding clamp is used to keep the DNA polymerase firmly in place during replication
 A clamp loader is used to hydrolyse an ATP each time it locks the sliding clamp in place
 For the leading strand, it occurs once per cycle
 For the lagging strand, since the clamp must be removed and added for every
fragment, the loader must use much more ATP



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