Module 2 - DNA replication Flashcards
DNA Topoisomerase I
“Nicks” a single strand which allows the other strand to move through the gap. The single strand then rejoins after the other strand has moved through
DNA Topoisomerase II
“Nicks” both strands and then the DNA strands uncoil themselves
Helicase
Helicase breaks the hydrogen bonds between bases.
This is different to Topoisomerase which just relieves the stress in the supercoiling.
Replication fork
Point where DNA is being separated and synthesised.
Exonuclease activity
Degrading of DNA on the outside of DNA (i.e. at the end of Okazaki fragments), it occurs in two ways:
3’ to 5’ exonuclease activity
5’ to 3’ exonuclease activity
3’ to 5’ exonuclease activity
Used to go back and remove any incorrect nucleotides.
This is why there are rarely any mistakes in DNA replication - proofreading occurs.
5’ to 3’ exonuclease activity
tbc
Leading strand replication (Bacteria)
Primase creates a primer that is around 4 to 15 nucleotides in length and DNA polymerase III creates a new strand
Leading strand replication (Eukaryotes)
DNA polymerase alpha adds around 20 nucleotides to the primer and then DNA polymerase delta continues the replication
DNA Polymerase I (B)
Subunits:
5’ to 3’ exonuclease activity:
3’ to 5’ exonuclease activity:
Function:
One
Yes
Yes
DNA repair, replication
DNA Polymerase III (B)
Subunits:
5’ to 3’ exonuclease activity:
3’ to 5’ exonuclease activity:
Function:
At least ten
No
Yes
Main replicating enzyme
DNA Polymerase alpha (E)
Subunits:
5’ to 3’ exonuclease activity:
3’ to 5’ exonuclease activity:
Function:
Four
No
No
Priming during replication
DNA Polymerase delta (E)
Subunits:
5’ to 3’ exonuclease activity:
3’ to 5’ exonuclease activity:
Function:
Two/three
No
Yes
Main replicating enzyme
Okazaki fragments in bacteria
At the replication fork, fragments are made in the 5’ to 3’ direction.
As DNA Polymerase III reaches the following primer for the following Okazaki fragment, it must detach as it has no 5’ to 3’ exonuclease activity. DNA Polymerase then attaches and degrades the primer and makes it into DNA.
DNA ligase then links both Okazaki fragments, forming one continuous DNA strand.
Okazaki fragments in eukaryotes
DNA Polymerase delta reaches the next primer and then, along with helicase to break the bonds, it moves the next primer and the first few nucleotides of the next Okazaki fragments out of the way.
The Okazaki fragment that has been lifted is still attached so FEN1 (an endonuclease) degrades the DNA so that all extra nucleotides are removed from the strand.
DNA ligase then forms a phosphodiester bond between the two nucleotides.
Endonuclease activity
The degrading of DNA within the DNA strand (i.e. the breaking of a primer and part of an Okazaki fragment that has been moved by DNA Polymerase delta and helicase)
The end replication problem
The final Okazaki fragment cannot be made
because its priming site would be after the end of the parent molecule.
Therefore, as replication occurs, DNA molecules become shorter and shorter.
Telomerase
In eukaryotes, a special enzyme called telomerase prevents the ends of chromosomes from being shortened by adding telomeres to the end of DNA molecules.
By adding the sequence TTAGGG several times, the final Okazaki fragment can now be primed as the primer attaches far enough away that DNA Polymerase III can create the last Okazaki fragment.
Tetrahymena thermophila
Single-cell ciliated organisms with 40,000 chromosomes need a lot of telomerase.
Telomeres in Tetrahymena have the sequence TTGGGG while we have TTAGGG
Senescence
The process of cells without Telomerase getting shorter and dying.
In culture, cells can divide only around 50 times before senescence occurs.
Telomerase in all cells?
Telomerase is only present in stem cells.
The reason is unclear. However, in cancer cells, telomerase is switched on and chromosomes are longer than usual. Possible explanation?
S phase of the cell cycle
This is when DNA replication occurs. At this point, modified nucleotides can be incorporated into DNA and then detected with fluorescently labelled antibodies.
This is how we visualise sites of DNA replication.
Heterochromatin
Highly condensed, gene-poor, and transcriptionally silent.
Although it is gene-poor, it still has a significant effect as it is used to keep stability within chromosomes.
Euchromatin
Less condensed, gene-rich, and more easily transcribed.
Euchromatin is very often under transcription.
