Mechanism of DNA Replication (DP IB Biology)

Directionality of DNA Polymerase

• Similar to transcription and translation, DNA replication must occur in the 5’ to 3’ direction

• DNA polymerase only works in a 5’ to 3’ direction, adding nucleotides to the 3’ end of a strand of nucleotides

• DNA nucleotides have a phosphate bonded to the 5’ carbon of the deoxyribose sugar

• When DNA polymerase adds a new nucleotide to extend the DNA strand, the 5’ phosphate group of the incoming DNA nucleotide bonds to the free 3’ -OH group on the growing strand

DNA nucleotide structure diagram

DNA nucleotides have a phosphate bonded to the 5’ carbon of the pentose sugar

5' and 3' ends of a DNA strand diagram

When DNA polymerase adds a new nucleotide, the 5’ phosphate group of the incoming nucleotide bonds to the free 3’ -OH group on the growing strand

The Leading & Lagging Strand

• Double-stranded DNA consists of two antiparallel strands 

  • This means that one strand runs from 5' to 3', while the other strand runs from 3' to 5'

• During DNA replication, the two strands are ‘unzipped’ and DNA polymerase moves along each template strand linking nucleotides together to form a new strand

  • Crucially, DNA polymerase can only add new nucleotides in a 5’ to 3’ direction

  • As the template strands are antiparallel, replication needs to proceed in opposite directions

• As the replication fork opens up in one direction only, each new strand is synthesised differently

  • The leading strand is made continuously, following the fork as it opens

  • The lagging strand is made discontinuously, in short fragments, away from the fork

    • These fragments are called Okazaki fragments

• As more template strand is exposed, new fragments are created

  • Okazaki fragments are later joined together by DNA ligase to form a continuous complementary DNA strand

• Before new DNA nucleotides can be added to the new DNA strand, first an RNA primer must be added to create a binding point for DNA polymerase III

  • The RNA primer only has to be added once on the leading strand but several are needed on the lagging strand to initiate each fragment

Difference between replication on the lagging and leading strands of DNA diagram

During DNA replication, synthesis of the leading strand is continuous but synthesis of the the lagging strand is discontinuous in small fragments (not all the enzymes involved are shown)

Enzymes in DNA Replication

• DNA replication is carried out by a complex system of enzymes working as a team

• Helicase unwinds the DNA double helix at the replication fork 
  

  • Helicase then causes the hydrogen bonds between the two strands to break so that they can separate

• Single-stranded binding proteins keep the separated strands apart whilst the template strand is copied

• DNA primase generates a short RNA primer on the template strands

  • Providing an initiation point for DNA polymerase III to add new nucleotides

• A number of polymerases are involved in DNA replication, each with different functions

  • Two of these polymerases are

    • DNA polymerase III, which starts replication next to the RNA primer linking nucleotides in a 5’ to 3’ direction to form a new strand

    • DNA polymerase I, which removes the RNA primers on the leading and lagging strands and replaces it with DNA

• DNA ligase joins up the Okazaki fragments by catalysing the formation of sugar-phosphate bonds

Proofreading Replicated DNA

• Each time a human cell replicates it requires 3 billion new base pairs to be synthesised in order to fully replicate the genome

• The copying process is not 100% perfect and mistakes do occur, these are called mutations

  • Mutations can be harmful to the functioning of the new cell and lead to diseases such as cancer

• In prokaryotes, in order to reduce mistakes during replication the enzyme DNA polymerase III acts as a proof-reader of the new daughter strand of DNA

  • It can recognise incorrect DNA nucleotides in the daughter strand

  • It reverses direction in order to remove the incorrect nucleotide from the 3' end of this strand

  • The correct nucleotide is then inserted and the polymerase III enzyme continues replication