Download DNA Replication

DNA replication (4 lectures) and cell-cycle (1 lecture)
Anindya Dutta
Presentations at the MICR811 toolkit site
Also at
http://www.cs.virginia.edu/~am3cp/DNARepl/
http://mexico.bioch.virginia.edu/DNARepl
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
G1
S
G2
M
Activity
Growth and increase in cell size
DNA synthesis
Post-DNA synthesis
Mitosis
Duration
10 hr
8 hr
5 hr
1 hr
DNA replication has two requirements that must be met:
1.
2.
DNA template
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
G1
S
G2
M
Activity
Growth and increase in cell size
DNA synthesis
Post-DNA synthesis
Mitosis
Duration
10 hr
8 hr
5 hr
1 hr
DNA replication has two requirements that must be met:
1.
2.
DNA template
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 singlestranded 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 discovery of DNA polymerase.
Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain
of deoxyribonucleic acid just like glycogen synthase elongates a chain of glycogen.
The enzymatic activity was unusual:
1) Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity
2) Needed a primer annealed to the template.
exonuclease
I
Wait a minute!
Either the polymerase
hypothesis was all
wrong,…… or there were
other DNA polymerases
in E. coli
that carried out DNA
synthesis in the polA
strains.
200
polA + (wild type)
0.4M
II
III
0.2M
100
+NEM
polA- (Cairns)
200
II
0.4M
III
0.2M
100
I
20
30
Fractions
40
Phosphate (M)
John Cairns mutated the
gene for DNA
polymerase, polA, and
the bacteria grew just
fine!
3H Thymidine incorporation (pmol)
600
Sub kDa
unit
Gene
Subassembly


130
27.5
|
| POL III CORE

dnaE
dnaQ
(mutD)
10

71
dnaX


'


47.5
35
33
15
12
dnaX

40.6
dnaN
|
|
|
|  COMPLEX
|
|
 CLAMP
DNA POL YMERASE III
5'-3' polymerase
5'-3'
exonuclease
3'-5'
exonuclease
ATP dependent
clamploader
processivity
factor
The end-replication problem
Solution to the end-replication problem
Telomerase
Telomeres contain arrays of DNA repeats
Group
Organism Telomeric repeat (5' to 3' toward the end)
Vertebrates
Human, mouse, Xenopus
TTAGGG
Filamentous
fungi
Neurospora
TTAGGG
Physarum, Didymium
Dictyostelium
TTAGGG
AG(1-8)
Tetrahymena, Glaucoma
Paramecium
Oxytricha, Stylonychia,
Euplotes
TTGGGG
TTGGG(T/G)
TTTTGGGG
Schizosaccharomyces pombe
TTAC(A)(C)G(1-8)
Slime molds
Ciliated
protozoa
Fission yeasts
Budding yeasts Saccharomyces cerevisiae
TGTGGGTGTGGTG
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
Looping the
lagging
strand to
make both
polymerases
move in the
same
direction
Sub kDa
unit
Gene
Subassembly


130
27.5
|
| POL III CORE

dnaE
dnaQ
(mutD)
10

71
dnaX


'


47.5
35
33
15
12
dnaX

40.6
dnaN
|
|
|
|  COMPLEX
|
|
 CLAMP
DNA POL YMERASE III
5'-3' polymerase
5'-3'
exonuclease
3'-5'
exonuclease
ATP dependent
clamploader
processivity
factor
Sub Gene
unit
Bacterial
Function
Eukaryotic

dnaE
|
DNA POL 

dnaQ
(mutD)
| POL III
CORE
|
5'-3'
polymerase
3'-5'
exonuclease
5'-3'
exonuclease
|
|
|
COMPLEX
|
|
ATP
dependent
clamploader


dnaX


'
dnaX



dnaN
 CLAMP
processivity
factor
DNA POL 
Fen1
RF-C
PCNA
CONSERVATION FROM PROKARYOTES TO
EUKARYOTES
Which polymerase is processive?
P
POL
dNTP
Challenge with vast excess of cold
primer-template
Gel electrophoresis of products
Challenge -
+
POL-X
-
+
POL-Y
POLIII,  subunit
PCNA
Clamp loaders hydrolyze ATP to load clamp
Clamp-loader
ATP
ATP
Clamp
How does one prove
that the clamp ring
is opened during
loading?
ATP
ADP
+ PP i
3‘OH
Structure of a DNA polymerase (gp43 from phage RB69)
Side view:
Polymerase active site
Top view with
template-primer:
Polymerase site
And
proofreading site
Topoisomerases relax DNA by changing the DNA
linking number
* Topoisomerases II change the linking number in steps of 2 by
passing both strands of double-stranded DNA through a break.
* Eukaryotic topoisomerases isolated to date only relax
supercoiled DNA, while prokaryotic topoisomerases (gyrases)
can, given ATP, add supercoils.
* TopoII releases catenated daughter molecules at the end of
replication. Inhibitors like etoposide are used in chemotherapy.
* Topoisomerases I change the linking number in steps of 1. They pass a
single DNA strand through a nick.Topoisomerase I is a protein of the
metaphase chromosome scaffold.
* In interphase, topoisomerase is bound to the nuclear matrix.
* The DNA replication machinery also appears bound to the matrix.
* Inhibitor (camptothecin) also used in chemotherapy.
Topoisomerase action can be divided into three steps:
nicking (1), strand passage (2); resealing (3).
5‘ end of DNA in gate
segment is covalently
linked to the OH of
tyrosine in the active
site of topo.
Cycle of topoisomerase activity inferred from structure
1
2
4
3
How would you test that the subunits have to open at the lower end to release the T segment?