Okazaki Fragments (only lecture notes, need to do further reading!)
- Reji & Tuneko Okazaki (1968)
- Conducted pulse-chase experiments
- Involved exposing replicating DNA to short "pulse" of radio-labelled nucleotides
- Then varying the length of time that the cells would be exposed to non-labelled nucleotides "chase"
- Okazaki determined that not all DNA synthesis was a smooth continous process
- For the lagging strand, fragments of DNA were synthesised discretely and assembled later on.
Okazaki's procedure
- Exposed replicating DNA to short pulse (5 s) of 3H radioactive nucleotides, followed by addition of excess of normal cold (non-radioactive) nucleotides.
- Resulted in label present only in DNA that synthesised during the short period of the pulse.
- DNA then isolates and strands separated in alkaline solution.
- Various pieced of DNA were sorted out by size using a "sucrose gradient" in an ultracentrifuge.
- Bigger pieces of DNA precipitated more rapidly than smaller pieces.
- Label occurred on 2 size of DNA, one very long and other on small fragments of 1000-2000 nucleotides long.
- So.. were the smaller fragments artificially induced breakdown products of normally large pieces?
- Answer... NO!
- Okazaki extended length of exposure pulse to 30sec, a far greater fraction of total label ended up in long DNA strands (similar results obtained in period of "cold chase" was prolonged.
- Therefore fragments existed only temporarily and soon became incorporated into the growing DNA strands.
DNA Polymerase I removes/replaces RNA primer
- Because of its 5' to 3' exonuclease activity, DNA polymerase I removes RNA primers and fills the gaps between okazaki with DNA.
Okazaki fragments joined by DNA ligase
- DNA ligase seals nicks in phosphodiester backbone of DNA where 3'-OH is next to a 5'-phosphate group.
- There are 2 classes of DNA ligases
- NAD+ as cofactor (E.coli)
- ATP as cofactor (T4 DNA ligase, eukaryotic)
- Joins okazaki fragments together making lagging strand continous covalently linked chain
- Mutants that were ligase-negative (lack a functional ligase) failed to show the pulse-chase assembled into large fragments!
Summary: DNA Replication
•DNA strands separate at origin of replication creating 2 replication forks
•Primers initiate synthesis. Leading strand replication proceeds in direction of replication fork
•1st Okazaki fragment is made in opposite direction for lagging strand
•Leading strand elongates and 2nd Okazaki fragment made
•3rd Okazaki fragment made and 1st & 2nd are connected together via DNA ligase
•DNA strands separate at origin of replication creating 2 replication forks
•Primers initiate synthesis. Leading strand replication proceeds in direction of replication fork
•1st Okazaki fragment is made in opposite direction for lagging strand
•Leading strand elongates and 2nd Okazaki fragment made
•3rd Okazaki fragment made and 1st & 2nd are connected together via DNA ligase
References:
- http://highered.mheducation.com/olc/dl/120076/micro04.swf --> good animation!
- http://www.hhmi.org/biointeractive/dna-replication-advanced-detail --> good video!
- Books, Molecular biology of the cell, 6th ed and Biochemistry, Berg, internation ed