REPLICATION

>Replication is the essential process occurring at the time of S phase of cell cycle

5 3

Identical base sequences

Figure 11.1 Watson/Crick proposed mechanism of DNA replication

RNA primer DNA polymerase cannot start DNA synthesis without a primer. Even on the lagging stand, each Okazaki fragment requires an RNA primer before DNA synthesis can start. The primer used in each case is a short (approximately five nucleotides long) piece of RNA and is synthesized by an RNA polymerase called primase (Fig. 4a). Primase can make RNA directly on the single-stranded DNA template because, like all RNA polymerases, it does not require a primer to begin synthesis. The RNA primer made by primase (Fig. 4b) is then extended by DNA polymerase III (Fig. 4c). DNA polymerase III synthesizes DNA for

 DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork DNA gyrase travels ahead of the helicase and alleviates these supercoils Single-strand binding proteins bind to the separated DNA strands to keep them apart Then short (10 to 12 nucleotides) RNA primers are synthesized b DNA primase.
These short strands start or prime DNA synthesis

both the leading and lagging strand. After DNA synthesis by DNA polymerase III, DNA polymerase I uses its 5 3 exonuclease activity to remove the RNA primer and then fills the gap with new DNA (Fig. 4e and f). DNA polymerase III cannot carry out this task because it lacks the 5 3 activity of DNA polymerase I. Finally, DNA ligase joins the ends of the DNA fragments together

Figure 11.8 Schematic representation of DNA Polymerase III

Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA

The DNA template is a double helix with each strand wound tightly around the other and hence the two strands must be unwound during replication. How is this unwinding problem solved? A DNA helicase is used to unwind the double helix (using ATP as energy source) and SSB (singlestranded DNA-binding) protein prevent the single-stranded regions from basepairing again so that each of the two DNA strands is accessible for replication.

Direction of synthesis on leading strand

3 5 3

5 3 5

Figure 11.7 Two dimensional view of a replication fork

Figure 11.13 Three Dimensional view of Replication Fork

Direction of synthesis of leading strand

Direction of synthesis Of lagging strand

Direction of fork movement

In principle, for a replication fork to move along a piece of DNA, the DNA helix would need to unwind ahead of it, causing the DNA to rotate rapidly. However, the bacterial chromosome is circular and so there are no ends to rotate. The solution to the problem is that an enzyme called topoisomerase I breaks a phosphodiester bond in one DNA strand (a single-strand break) a small distance ahead of the fork, allowing the DNA to rotate freely (swivel) around the other (intact) strand. The phosphodiester bond is then re-formed by the topoisomerase.

DNA Replication
Origins of replication 1. Replication Forks hundreds of Y-shaped Forks: regions of replicating DNA molecules where new strands are growing.
3

5 3

Parental DNA Molecule Replication Fork

DNA Replication
2. Leading Strand: synthesized as a Strand single polymer in the 5 to 3 direction direction.

3 5 Nucleotides

DNA Polymerase

RNA Primer

DNA Replication
Strand Separation Separation: 1. Helicase: enzyme which catalyze the Helicase unwinding and separation (breaking HBonds) of the parental double helix. 2. Single-Strand Binding Proteins: SingleProteins proteins which attach and help keep the separated strands apart.

DNA Replication
Origins of replication 2. Replication Bubbles Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). (Eukaryotes) Single replication fork (bacteria). b.

Bubbles

Bubbles

DNA Replication
5. DNA ligase: a linking enzyme that ligase catalyzes the formation of a covalent bond from the 3 to 5 end of joining stands.
Example: joining two Okazaki fragments together.
DNA ligase
5 Okazaki Fragment 1 Okazaki Fragment 2 3

3 Lagging Strand

DNA Replication
4. Okazaki Fragments: series of short Fragments segments on the lagging strand.

Okazaki Fragment RNA Primer

DNA Polymerase

3 5

3 Lagging Strand

DNA Replication
3. Lagging Strand: also synthesized in Strand 5 to 3 direction but discontinuously direction, overall direction of replication.
5 3

the against

Leading Strand

3 5

DNA Polymerase
5 3 Lagging Strand

RNA Primer
3 5

DNA Replication
Synthesis of the new DNA Strands: 1. DNA Polymerase: with a RNA primer in Polymerase place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5 to 3 direction. direction
5 3 5 Nucleotide

DNA Polymerase

RNA Primer