DNA Replication and DNA Repair
(BOOK)
Chapter 5 pp 254-276
DNA Replication, Repair and Recombination
The initiation and completion of DNA replication in chromosomes
To begin DNA replication, the double helix must first be opened up and the two strands separated.
This begins by special initiator proteins which bind to the helix and pry the two strands apart. The
position where the DNA helix is first opened is called the replication origin. DNA rich in A—T base
pairs are typically found at replication origins because these bonds are easier to break than the C—G
bonds (A—T has two hydrogen bonds and C—G three).
Origin of DNA replication in bacteria
In bacteria with circular DNA the two replication forks
assembled at the replication origin proceed in opposite
direction until they meet each other again about halfway
around the chromosome. In E. coli the interaction of the
initiator protein with the replication origin is carefully
regulated because this is the only point it can control DNA
replication.
After replication is initiated the initiator protein is
inactivated by hydrolysis of its bound ATP, and the
replication origin experiences a ‘’refractory period.’’ This is
causes by a delay in the delation of newly incorporated A
nucleotides in the origin.
Origin of DNA replication in eukaryotes
Because the eukaryotic genome is much bigger than the
bacterial genome, there must be another process to
complete DNA replication in about the same time.
Therefore, many forks, belonging to separate replication
bubbles are moving simultaneously on each eukaryotic chromosome.
Experiments have shown a couple of things:
- 30.000 – 50.000 replication origins are used each time a human cell divides
- The human genome has many more potential origins than the numbers mentioned above
(the excess origins also functions as backup)
- Different cell types use different sets of origins
- Replication forks are formed in pairs and create a replication bubble as they move in
opposite direction
In bacteria DNA is almost continuously replicated, in eukaryotes in contrast this happens only during
one part of the cell cycle. DNA replication only happens during the DNA synthesis phase (S phase).
In mammalian cells, the S phase lasts for about 8 hours instead of the expected 1 hour (given the
amount of replication forks and speed). This is because not all replication origins are activated at the
same time. They are activated in clusters of about 50 adjacent replication origins.
, The single budding yeast S. cerevisiae
In the S. cerevisiae most DNA sequences that can serve as an
replication origin are found to contain:
1. A binding site for a large multisubunit initiator protein
called ORC (origin recognition complex)
2. A stretch of DNA that is rich in As and Ts and therefore
easy to melt
3. At least one binding site for proteins that facilitate ORC
binding
But with so many places to begin replication you have to be sure
to copy all the DNA only once. This is done by the loading and
activating of the replicative helicase. The replicative helicases are
loaded onto DNA next to ORC to create a prereplicative complex.
Then specialized kinases come into play to activate the helicases.
These protein kinases also prevent assembly of new prereplicative
complexes until the next M phase resets the entire cycle. This is
done by phosphorylating the ORCs.
Compared to budding yeast, the determinants of replication in
other eukaryotes have been difficult to discover. What has been
found is that a human ORC that is very similar to the yeast ORC
binds to the origin of replication and initiates DNA replication in
humans.
New nucleosomes are assembled behind the replication fork
An eukaryotic cell requires a large amount of new histone protein to make the new nucleosomes in
each cell cycle. For this reason, most eukaryotic organisms possess multiple copies of the gene for
each histone. Histones are mainly synthesised in the S phase. The tight linkage between DNA
synthesis and histone synthesis appears to reflect a feedback mechanism that monitors the level of
free histone to ensure that the amount of histone made exactly matches the amount of new DNA
synthesised.
As a replication fork passes through chromatin, the histones are transiently displaced leaving about
600 nucleotide pairs on non-nucleosomal DNA in its wake. When a nucleosomes is traversed by a
replication fork, the histone ocamer appears to be broken up into an H3-H4-tetramer and two H2A-
H2B dimers. The H3-H4-tetramer is reused, but the H2A-H2B dimers are released and replaced by
new ones. The orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers behind a
replication fork requires histone chaperones.
(BOOK)
Chapter 5 pp 254-276
DNA Replication, Repair and Recombination
The initiation and completion of DNA replication in chromosomes
To begin DNA replication, the double helix must first be opened up and the two strands separated.
This begins by special initiator proteins which bind to the helix and pry the two strands apart. The
position where the DNA helix is first opened is called the replication origin. DNA rich in A—T base
pairs are typically found at replication origins because these bonds are easier to break than the C—G
bonds (A—T has two hydrogen bonds and C—G three).
Origin of DNA replication in bacteria
In bacteria with circular DNA the two replication forks
assembled at the replication origin proceed in opposite
direction until they meet each other again about halfway
around the chromosome. In E. coli the interaction of the
initiator protein with the replication origin is carefully
regulated because this is the only point it can control DNA
replication.
After replication is initiated the initiator protein is
inactivated by hydrolysis of its bound ATP, and the
replication origin experiences a ‘’refractory period.’’ This is
causes by a delay in the delation of newly incorporated A
nucleotides in the origin.
Origin of DNA replication in eukaryotes
Because the eukaryotic genome is much bigger than the
bacterial genome, there must be another process to
complete DNA replication in about the same time.
Therefore, many forks, belonging to separate replication
bubbles are moving simultaneously on each eukaryotic chromosome.
Experiments have shown a couple of things:
- 30.000 – 50.000 replication origins are used each time a human cell divides
- The human genome has many more potential origins than the numbers mentioned above
(the excess origins also functions as backup)
- Different cell types use different sets of origins
- Replication forks are formed in pairs and create a replication bubble as they move in
opposite direction
In bacteria DNA is almost continuously replicated, in eukaryotes in contrast this happens only during
one part of the cell cycle. DNA replication only happens during the DNA synthesis phase (S phase).
In mammalian cells, the S phase lasts for about 8 hours instead of the expected 1 hour (given the
amount of replication forks and speed). This is because not all replication origins are activated at the
same time. They are activated in clusters of about 50 adjacent replication origins.
, The single budding yeast S. cerevisiae
In the S. cerevisiae most DNA sequences that can serve as an
replication origin are found to contain:
1. A binding site for a large multisubunit initiator protein
called ORC (origin recognition complex)
2. A stretch of DNA that is rich in As and Ts and therefore
easy to melt
3. At least one binding site for proteins that facilitate ORC
binding
But with so many places to begin replication you have to be sure
to copy all the DNA only once. This is done by the loading and
activating of the replicative helicase. The replicative helicases are
loaded onto DNA next to ORC to create a prereplicative complex.
Then specialized kinases come into play to activate the helicases.
These protein kinases also prevent assembly of new prereplicative
complexes until the next M phase resets the entire cycle. This is
done by phosphorylating the ORCs.
Compared to budding yeast, the determinants of replication in
other eukaryotes have been difficult to discover. What has been
found is that a human ORC that is very similar to the yeast ORC
binds to the origin of replication and initiates DNA replication in
humans.
New nucleosomes are assembled behind the replication fork
An eukaryotic cell requires a large amount of new histone protein to make the new nucleosomes in
each cell cycle. For this reason, most eukaryotic organisms possess multiple copies of the gene for
each histone. Histones are mainly synthesised in the S phase. The tight linkage between DNA
synthesis and histone synthesis appears to reflect a feedback mechanism that monitors the level of
free histone to ensure that the amount of histone made exactly matches the amount of new DNA
synthesised.
As a replication fork passes through chromatin, the histones are transiently displaced leaving about
600 nucleotide pairs on non-nucleosomal DNA in its wake. When a nucleosomes is traversed by a
replication fork, the histone ocamer appears to be broken up into an H3-H4-tetramer and two H2A-
H2B dimers. The H3-H4-tetramer is reused, but the H2A-H2B dimers are released and replaced by
new ones. The orderly and rapid addition of new H3-H4 tetramers and H2A-H2B dimers behind a
replication fork requires histone chaperones.
