DNA metabolism and cell cycle control
Lecture 1: DNA RRR
Replication, repair, recombination
Replication
- DNA polymerases
- The beta-clamp its loader and partners
- Replication origins
- Semi-conservative DNA replication mechanism
- Lagging strand synthesis
- Origin licensing
- DNA polymerases – (mitochondrial homeostasis and
aging)
- Telomerase – the T-loop (Senescence – cellular aging
If a mistake is introduced, descendant cells will carry it and their fitness may be affected.
Cancer impacts on longevity an is caused by mutations in the genetic code that deregulate
cell proliferation control.
Deoxy: 2’ end doesn’t have an oxygen
DNA replication always begins with a primer
- Contrary to RNA polymerases, cellular DNA polymerses cannot initiate DNA synthesis in the
absence of a primer
- Priming is performed by a specialized polymerase which is called primae
- Primase starts by making a 10-20 bp RNA primer, the 3’OH end of this primer is alter
elongated by a DNA polymerase that was loaded onto the primer-bound DNA template.
- Every Okazaki fragment on the lagging strand is primed by primase.
Chromosomal replication fork migration is coupled to repeated initiation steps on the lagging strand.
- Okazaki fragments must be started by
primase
- The RNA primers are removed by the 5’-3’
exonuclease domain of DNA polymerase
the remaining nick is repaired by ligase.
Elongating- chromosome replicating – DNA polymerases faces 3 choices
1. Carry on with DNA polymerization (5’ >>3’)
2. Reverse polymerization using (5’ >>3’) proofreading exonuclease activity
3. Release the template.
Choosing 1 is favoured. This is an intrinsic property, built into the 3D architecture of the polymerase
on a DNA template with Mg2+ and deoxynucleotides (dNTPs)
The switch from 1 to 2 is induced by detection of aberrant base pair geometries within the growing
DNA binding site of the polymerase.
Choice 3 is inhibited by DNA clamps.
Very high polymerisation processivity is achieved through topological trapping of DNA by circular
protein complexes
, - PCNA must be loaded onto DNA by a clamp loading complex which is called replication factor
c (RFC)
- multiple isoforms of RCF exist.
Because PCNA is a homotrimer, one PCNA ring can simultaneously bind multiple PCNA partners
This permits coordination of DNA replication/associated molecular signalling.
During chromosome duplication all the DNA must be replicated (in eukaryotic cells they must be
replicated once) *
- Bi-directional DNA replication >> bidirectional growth of both strands from one origin
- Helicase unwinds origin
- Leading strand synthesis starts on ssDNA.
- Lagging strand synthesis starts on ssDNA
- More Okazaki fragment priming
- Okazaki fragment ligation
- The two forks are moving away from each other
- Eventually each fork meets another fork and
terminates DNA synthesis
Eukaryotic DNA replication enzymology
- Resembles prokaryote enzymology
- Eukaryotic DNA pol >> bacterial DNA pol III
homologues for the lagging and for the leading strand
- ORC (origin of replication complex) marks origins
throughout the cell cycle
- MCM complex is the helicase that unwinds DNA in
front of the fork
- Topoisomerases relieve torsional stress caused by DNA
unwinding
- RPA (replication protein A) binds ssDNA
- RFC (replication factor C) loads PCNA on DNA
- PCNA (proliferating cell nuclear antigen) = dsDNA clmap that tethers the DNA polymerase to
its template, as well as cyclins, CDKs and DNA repair and recombination factors.
*all the DNA is duplicated once and only once >> during mitosis the two (sister) copies are segregated
to the two daughter cells
- there is an origin licensing mechanism that ensures that any origin of replication fires only once per
cell cycle, or not at all.
How do you avoid double duplication >> “ticket” to be able to duplicate.
- Eukaryotic replication origins are bound by the ORC complex throughout the cell division
cycle.
Lecture 1: DNA RRR
Replication, repair, recombination
Replication
- DNA polymerases
- The beta-clamp its loader and partners
- Replication origins
- Semi-conservative DNA replication mechanism
- Lagging strand synthesis
- Origin licensing
- DNA polymerases – (mitochondrial homeostasis and
aging)
- Telomerase – the T-loop (Senescence – cellular aging
If a mistake is introduced, descendant cells will carry it and their fitness may be affected.
Cancer impacts on longevity an is caused by mutations in the genetic code that deregulate
cell proliferation control.
Deoxy: 2’ end doesn’t have an oxygen
DNA replication always begins with a primer
- Contrary to RNA polymerases, cellular DNA polymerses cannot initiate DNA synthesis in the
absence of a primer
- Priming is performed by a specialized polymerase which is called primae
- Primase starts by making a 10-20 bp RNA primer, the 3’OH end of this primer is alter
elongated by a DNA polymerase that was loaded onto the primer-bound DNA template.
- Every Okazaki fragment on the lagging strand is primed by primase.
Chromosomal replication fork migration is coupled to repeated initiation steps on the lagging strand.
- Okazaki fragments must be started by
primase
- The RNA primers are removed by the 5’-3’
exonuclease domain of DNA polymerase
the remaining nick is repaired by ligase.
Elongating- chromosome replicating – DNA polymerases faces 3 choices
1. Carry on with DNA polymerization (5’ >>3’)
2. Reverse polymerization using (5’ >>3’) proofreading exonuclease activity
3. Release the template.
Choosing 1 is favoured. This is an intrinsic property, built into the 3D architecture of the polymerase
on a DNA template with Mg2+ and deoxynucleotides (dNTPs)
The switch from 1 to 2 is induced by detection of aberrant base pair geometries within the growing
DNA binding site of the polymerase.
Choice 3 is inhibited by DNA clamps.
Very high polymerisation processivity is achieved through topological trapping of DNA by circular
protein complexes
, - PCNA must be loaded onto DNA by a clamp loading complex which is called replication factor
c (RFC)
- multiple isoforms of RCF exist.
Because PCNA is a homotrimer, one PCNA ring can simultaneously bind multiple PCNA partners
This permits coordination of DNA replication/associated molecular signalling.
During chromosome duplication all the DNA must be replicated (in eukaryotic cells they must be
replicated once) *
- Bi-directional DNA replication >> bidirectional growth of both strands from one origin
- Helicase unwinds origin
- Leading strand synthesis starts on ssDNA.
- Lagging strand synthesis starts on ssDNA
- More Okazaki fragment priming
- Okazaki fragment ligation
- The two forks are moving away from each other
- Eventually each fork meets another fork and
terminates DNA synthesis
Eukaryotic DNA replication enzymology
- Resembles prokaryote enzymology
- Eukaryotic DNA pol >> bacterial DNA pol III
homologues for the lagging and for the leading strand
- ORC (origin of replication complex) marks origins
throughout the cell cycle
- MCM complex is the helicase that unwinds DNA in
front of the fork
- Topoisomerases relieve torsional stress caused by DNA
unwinding
- RPA (replication protein A) binds ssDNA
- RFC (replication factor C) loads PCNA on DNA
- PCNA (proliferating cell nuclear antigen) = dsDNA clmap that tethers the DNA polymerase to
its template, as well as cyclins, CDKs and DNA repair and recombination factors.
*all the DNA is duplicated once and only once >> during mitosis the two (sister) copies are segregated
to the two daughter cells
- there is an origin licensing mechanism that ensures that any origin of replication fires only once per
cell cycle, or not at all.
How do you avoid double duplication >> “ticket” to be able to duplicate.
- Eukaryotic replication origins are bound by the ORC complex throughout the cell division
cycle.
