Biochemistry and Molecular Biology II – Lecture 8: DNA Replication, Repair and
Recombination – Lecture by Colin Logie
15-05-2018
- Polymerases always add to the 3’ end ( 5’ 3’ end)
- DNA replication always begins with a primer (added by primase)
- The RNA-primers are removed by the 5’ 3’ end exonuclease domain of DNA
polymerase I
- The remaining ‘nicks’ between okazaki-fragments are repaired by ligase.
Elongating DNA polymerase faces 3 choices:
1. Carry on with chain elongation (5’ 3’ end)
2. Reverse polymerization using its 3’ 5’ end exonuclease activity
3. Release the template
* Mode 1 is favored. This is an intrinsic property built into the 3D architecture of the polymerase.
*The choice to switch from 1 to 2 is induced by detection of aberrant base pair geometries within
the double strand DNA binding site.
* Mode 3 is inhibited by DNA clamps
Very high polymerization processivity is achieved through topological trapping of DNA, by
circular protein complexes:
In eukaryotes this trimeric DNA clamp is called PCNA (Proliferating Cell Nuclear Antigen),
preventing the template from being released.
PCNA is loaded onto DNA by a clamp-loading-complex which is called RFC (Replication Factor
C).
In prokaryotes this dimeric DNA-clamp is called the B-clamp.
- Multiple isoforms of RFC exist.
- For DNA replication, RFC assembles PCNA onto ssDNA decorated with a primer
synthesized by primase.
, - The DNA-clamps PCNA and B-clamp make sure that a higher polymerization
processivity is achieved.
*Because PCNA is a homotrimer, one PCNA ring can simultaneously bind multiple PCNA
partners.
this permits coordination of DNA replication-associated molecular signaling.
*All different factors know where to go to, because of their binding ability to PCNA;
!!The many partners of PCNA indicate that next to being a DNA polymerase processivity
factor, PCNA also has a central role in signaling chromosome replication fork status and in
coordinating molecular events that lead to DNA damage repair and cell cycle progression
control.
General chromosome replication mechanisms:
*In eukaryotes there are 4 replication forks per replication origin.
- Telomeres, telomerase and tumor suppression
What must happen during chromosome duplication?
All DNA must be replicated only once.
This is done by bi-directional DNA replication;
DNA-helicase unwinds the DNA strands, creating a ‘fork’ where replication can occur both left
and right, with use of primers and DNA-polymerases.
How to show this in an experimental setting?
- By conjugation of products you can visualize several products visualize bi-directional
replication, with use of fluorophores.
*There is one problem with bidirectional replication:
Both the 5’ 3’ and 3’ 5’ strands must be replicated by both forks, whilst DNA polymerases
only work in the 5’ 3’ end direction.
- In nature this is solved by discontinuous DNA-synthesis on the lagging strand
resulting in Okazaki-fragments as intermediate products.
!! When two replication forks meet each other, DNA-synthesis is terminated intrinsic
property.
Recombination – Lecture by Colin Logie
15-05-2018
- Polymerases always add to the 3’ end ( 5’ 3’ end)
- DNA replication always begins with a primer (added by primase)
- The RNA-primers are removed by the 5’ 3’ end exonuclease domain of DNA
polymerase I
- The remaining ‘nicks’ between okazaki-fragments are repaired by ligase.
Elongating DNA polymerase faces 3 choices:
1. Carry on with chain elongation (5’ 3’ end)
2. Reverse polymerization using its 3’ 5’ end exonuclease activity
3. Release the template
* Mode 1 is favored. This is an intrinsic property built into the 3D architecture of the polymerase.
*The choice to switch from 1 to 2 is induced by detection of aberrant base pair geometries within
the double strand DNA binding site.
* Mode 3 is inhibited by DNA clamps
Very high polymerization processivity is achieved through topological trapping of DNA, by
circular protein complexes:
In eukaryotes this trimeric DNA clamp is called PCNA (Proliferating Cell Nuclear Antigen),
preventing the template from being released.
PCNA is loaded onto DNA by a clamp-loading-complex which is called RFC (Replication Factor
C).
In prokaryotes this dimeric DNA-clamp is called the B-clamp.
- Multiple isoforms of RFC exist.
- For DNA replication, RFC assembles PCNA onto ssDNA decorated with a primer
synthesized by primase.
, - The DNA-clamps PCNA and B-clamp make sure that a higher polymerization
processivity is achieved.
*Because PCNA is a homotrimer, one PCNA ring can simultaneously bind multiple PCNA
partners.
this permits coordination of DNA replication-associated molecular signaling.
*All different factors know where to go to, because of their binding ability to PCNA;
!!The many partners of PCNA indicate that next to being a DNA polymerase processivity
factor, PCNA also has a central role in signaling chromosome replication fork status and in
coordinating molecular events that lead to DNA damage repair and cell cycle progression
control.
General chromosome replication mechanisms:
*In eukaryotes there are 4 replication forks per replication origin.
- Telomeres, telomerase and tumor suppression
What must happen during chromosome duplication?
All DNA must be replicated only once.
This is done by bi-directional DNA replication;
DNA-helicase unwinds the DNA strands, creating a ‘fork’ where replication can occur both left
and right, with use of primers and DNA-polymerases.
How to show this in an experimental setting?
- By conjugation of products you can visualize several products visualize bi-directional
replication, with use of fluorophores.
*There is one problem with bidirectional replication:
Both the 5’ 3’ and 3’ 5’ strands must be replicated by both forks, whilst DNA polymerases
only work in the 5’ 3’ end direction.
- In nature this is solved by discontinuous DNA-synthesis on the lagging strand
resulting in Okazaki-fragments as intermediate products.
!! When two replication forks meet each other, DNA-synthesis is terminated intrinsic
property.
