Electron Microscopy of replicating DNA reveals
replicating bubbles,How does one prove
bidirectional fork movement?
Pulse with radiolabeled nucleotide; chase with cold
nucleotide,Then do autoradiography
DNA Replication
DNA replication is semi-conservative,one strand serves as the template for the second strand,
Furthermore,DNA replication only occurs at a specific step in the cell cycle,
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle,
Stage Activity Duration
G1 Growth and increase in cell size 10 hr
S DNA synthesis 8 hr
G2 Post-DNA synthesis 5 hr
M Mitosis 1 hr
DNA replication has two requirements that must be met,
1,DNA template
2,Free 3' -OH group
DNA Replication
DNA replication is semi-conservative,one strand serves as the template for the second strand,
Furthermore,DNA replication only occurs at a specific step in the cell cycle,
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle,
Stage Activity Duration
G1 Growth and increase in cell size 10 hr
S DNA synthesis 8 hr
G2 Post-DNA synthesis 5 hr
M Mitosis 1 hr
DNA replication has two requirements that must be met,
1,DNA template
2,Free 3' -OH group
Proteins of DNA Replication
DNA exists in the nucleus as a condensed,compact structure,To prepare DNA for replication,
a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule,
These proteins are required because DNA must be single-stranded before replication can
proceed,
1,DNA Helicases - These proteins bind to the double stranded DNA and stimulate the
separation of the two strands,
2,DNA single-stranded binding proteins - These proteins bind to the DNA as a
tetramer and stabilize the single-stranded structure that is generated by the action of the
helicases,Replication is 100 times faster when these proteins are attached to the single-
stranded DNA,
3,DNA Topoisomerase - This enzyme catalyzes the formation of negative supercoils that is
thought to aid with the unwinding process,
In addition to these proteins,several other enzymes are involved in bacterial DNA replication,
4,DNA Polymerase - DNA Polymerase I (Pol I) was the first enzyme discovered with
polymerase activity,and it is the best characterized enzyme,Although this was the first
enzyme to be discovered that had the required polymerase activities,it is not the primary
enzyme involved with bacterial DNA replication,That enzyme is DNA Polymerase III (Pol III),
Three activities are associated with DNA polymerase I;
* 5' to 3' elongation (polymerase activity)
* 3' to 5' exonuclease (proof-reading activity)
* 5' to 3' exonuclease (repair activity)
The second two activities of DNA Pol I are important for replication,but DNA Polymerase III
(Pol III) is the enzyme that performs the 5'-3' polymerase function,
5,Primase - The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that
are synthesized at the initiation sites by these enzymes,
6,DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed
and synthesis proceeds in a discontinuous manner on the lagging strand,The final replication
product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage
between 3'-hydroxyl and 5'-phosphate groups.
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand,
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
RNA primed DNA replication
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand,
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand.
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
Removal of RNA primers and filling of gaps
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand.
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
ATP is an integral part of the ligation reaction
The end-replication problem
Group Organism Telomeric repeat (5' to 3' toward the end)
Vertebrates Human,mouse,Xenopus TTAGGG
Filamentous
fungi Neurospora TTAGGG
Slime molds Physarum,Didymium TTAGGG
Dictyostelium AG(1-8)
Ciliated
protozoa Tetrahymena,Glaucoma TTGGGG
Paramecium TTGGG(T/G)
Oxytricha,Stylonychia,TTTTGGGG
Euplotes
Fission yeasts Schizosaccharomyces pombe TTAC(A)(C)G(1-8)
Budding yeasts Saccharomyces cerevisiae TGTGGGTGTGGTG
Telomeres contain arrays of DNA repeats
Telomerase is
a reverse
transcriptase
together with
a template RNA
It is active in
germ cells,not
in somatic cells,
and is activated
in cancers
Finishing school for telomeres
replicating bubbles,How does one prove
bidirectional fork movement?
Pulse with radiolabeled nucleotide; chase with cold
nucleotide,Then do autoradiography
DNA Replication
DNA replication is semi-conservative,one strand serves as the template for the second strand,
Furthermore,DNA replication only occurs at a specific step in the cell cycle,
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle,
Stage Activity Duration
G1 Growth and increase in cell size 10 hr
S DNA synthesis 8 hr
G2 Post-DNA synthesis 5 hr
M Mitosis 1 hr
DNA replication has two requirements that must be met,
1,DNA template
2,Free 3' -OH group
DNA Replication
DNA replication is semi-conservative,one strand serves as the template for the second strand,
Furthermore,DNA replication only occurs at a specific step in the cell cycle,
The following table describes the cell cycle for a hypothetical cell with a 24 hr cycle,
Stage Activity Duration
G1 Growth and increase in cell size 10 hr
S DNA synthesis 8 hr
G2 Post-DNA synthesis 5 hr
M Mitosis 1 hr
DNA replication has two requirements that must be met,
1,DNA template
2,Free 3' -OH group
Proteins of DNA Replication
DNA exists in the nucleus as a condensed,compact structure,To prepare DNA for replication,
a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule,
These proteins are required because DNA must be single-stranded before replication can
proceed,
1,DNA Helicases - These proteins bind to the double stranded DNA and stimulate the
separation of the two strands,
2,DNA single-stranded binding proteins - These proteins bind to the DNA as a
tetramer and stabilize the single-stranded structure that is generated by the action of the
helicases,Replication is 100 times faster when these proteins are attached to the single-
stranded DNA,
3,DNA Topoisomerase - This enzyme catalyzes the formation of negative supercoils that is
thought to aid with the unwinding process,
In addition to these proteins,several other enzymes are involved in bacterial DNA replication,
4,DNA Polymerase - DNA Polymerase I (Pol I) was the first enzyme discovered with
polymerase activity,and it is the best characterized enzyme,Although this was the first
enzyme to be discovered that had the required polymerase activities,it is not the primary
enzyme involved with bacterial DNA replication,That enzyme is DNA Polymerase III (Pol III),
Three activities are associated with DNA polymerase I;
* 5' to 3' elongation (polymerase activity)
* 3' to 5' exonuclease (proof-reading activity)
* 5' to 3' exonuclease (repair activity)
The second two activities of DNA Pol I are important for replication,but DNA Polymerase III
(Pol III) is the enzyme that performs the 5'-3' polymerase function,
5,Primase - The requirement for a free 3' hydroxyl group is fulfilled by the RNA primers that
are synthesized at the initiation sites by these enzymes,
6,DNA Ligase - Nicks occur in the developing molecule because the RNA primer is removed
and synthesis proceeds in a discontinuous manner on the lagging strand,The final replication
product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage
between 3'-hydroxyl and 5'-phosphate groups.
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand,
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
RNA primed DNA replication
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand,
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand.
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
Removal of RNA primers and filling of gaps
A General Model for DNA Replication
1,The DNA molecule is unwound and prepared for synthesis by the action of DNA gyrase,DNA
helicase and the single-stranded DNA binding proteins,
2,A free 3'OH group is required for replication,but when the two chains separate no group of that
nature exists,RNA primers are synthesized,and the free 3'OH of the primer is used to begin replication,
3,The replication fork moves in one direction,but DNA replication only goes in the 5' to 3' direction.
This paradox is resolved by the use of Okazaki fragments,These are short,discontinuous replication
products that are produced off the lagging strand,This is in comparison to the continuous strand that is
made off the leading strand.
4,The final product does not have RNA stretches in it,These are removed by the 5' to 3' exonuclease
action of Polymerase I,
5,The final product does not have any gaps in the DNA that result from the removal of the RNA
primer,These are filled in by the 5’ to 3’ polymerase action of DNA Polymerase I,
6,DNA polymerase does not have the ability to form the final bond,This is done by the enzyme DNA
ligase,
ATP is an integral part of the ligation reaction
The end-replication problem
Group Organism Telomeric repeat (5' to 3' toward the end)
Vertebrates Human,mouse,Xenopus TTAGGG
Filamentous
fungi Neurospora TTAGGG
Slime molds Physarum,Didymium TTAGGG
Dictyostelium AG(1-8)
Ciliated
protozoa Tetrahymena,Glaucoma TTGGGG
Paramecium TTGGG(T/G)
Oxytricha,Stylonychia,TTTTGGGG
Euplotes
Fission yeasts Schizosaccharomyces pombe TTAC(A)(C)G(1-8)
Budding yeasts Saccharomyces cerevisiae TGTGGGTGTGGTG
Telomeres contain arrays of DNA repeats
Telomerase is
a reverse
transcriptase
together with
a template RNA
It is active in
germ cells,not
in somatic cells,
and is activated
in cancers
Finishing school for telomeres
