Lectures 19 and 20
DNA Replication is semiconservative
Meselson-Stahl experiment:
gradient centrifugation.
15N-labeling
and CsCl density
Chapter 12: DNA Replication and
Recombination
Problem set 3A: due at beginning of lecture on Monday,
Oct. 16th
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Overview of replication
General Features of Replication
Components required in vitro:
1. Enzyme: DNA polymerase
2. Template: DNA
3. deoxyribonucleotide triphosphates
(dNTP's = dATP, dCTP, dGTP, and dTTP)
4. Primer
1.
2.
3.
4.
5.
Synthesis is always in the 5'-->3' direction.
Starts at a specific point: "origin of replication" (=ori)
Is usually bidirectional (sometimes unidirectional)
Bacterial chromosome and plasmids each have one ori
Eukaryotic chrom's have many ori's. because they’re so long!
(Animation: Bidirectional Replication of DNA)
(Animation: Nucleotide Polymerization by DNA Polymerase.)
(To view animations go to the web site for the text book:
http://bcs.whfreeman.com/pierce2e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=
0&rau=0
This is the same site that is listed in the Course Logistics document from the
beginning of the course.
Click on: “Genetics in Motion: Interactive Animations”)
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Rolling circle replication, a type of replication done
by phiX174 and some other viruses
Linear chromosomes have multiple origins of replication.
3
Bacterial Replication Enzymes to Know:
Some enzymes to get to know--the big guys in bacterial replication.
A. Binds to origin and separates strands of DNA to initiate replication.
B. DNA gyrases (topoisomerases): reduce supercoiling of the DNA ahead
of replication fork by making, then resealing breaks in the DNA backbone.
C. Helicases: Denature DNA at replication fork.
D. Single-strand binding proteins : keep DNA single stranded.
E. Primase: an RNA polymerase that uses DNA as a template to
make RNA primer for DNA replication.
F. DNA polymerase III (at least 10 different polypeptide subunits):
elongates primers in the 5'--->3' direction, giving continuous
synthesis on the leading strand and discontinuous synthesis on the
lagging strand.
DNA Replication proteins in eukaryotic cells have similar functions
G. DNA polymerase I: digests RNA primers with its 5'--->3'
exonuclease activity and replaces with DNA.
H. DNA ligase: joins unlinked DNA strands by sealing nicks in the
sugar-phosphate backbone.
I. DNA polymerase II: involved in DNA repair.
4
Deoxyribonucleoside triphosphates are incorporated into the growing
DNA chain.
Animation: Coordination of Leading and Lagging-Strand Synthesis
5
Animation: Overview of Replication
6
7
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During replication DNA forms a loop to allow two DNA
polymerase III complexes to work together.
1. Accurate nucleotide selection
The fidelity of DNA replication is controlled at more than three levels.
Error rate: <1 nucleotide/billion!
2. DNA proofreading
A. Incorrect bases don’t fit well.
B. Many DNA polymerases pause if incorrect base is inserted.
C. If they have 3’Æ5’ exonuclease activity, they can cut off the incorrect base
D. The correct base can now be inserted.
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3. Mismatch repair: recognition of mismatched bases after
replication is complete.
Selective repair of the new strand, because
The old strand is recognized because some nucleotides on it are methylated.
(more on repair of DNA damage latter in course)
Telomerase: a ribonucleoprotein (RNA and protein) reverse transcriptase
Telomeres and the end replication problem.
1. Its RNA has sequence complementary to telomere sequence.
2. Uses its own RNA as template.
3. Adds additional telomeric repeats
to the ends of telomeres.
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1. Shortening of telomeres appears to contribute to cell death and aging.
A. germ-line cells: high telomerase activity
B. somatic cells: very low telomerase activity
2. Telomerase negative mice: premature aging after a # of generations.
3. Somatic cells engineered to have telomerase activity: divide indefinitely.
4. Telomerase activation is part of the development of most advanced cancers.
Recombination
I Involves proteins that:
1. nick DNA
2. help complementary strands base pair in new combos
3. resolve intermediate structures formed by further nicking and
ligation.
4. probably do small amounts of strand synthesis
II The Holliday model.
III The Double-strand-break repair model
(Animation: Mechanism of Homologous Recombination)
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The Holliday Model
1. Single strand nicking
4. Rotation of molecules
2. Single strand invasion leads to
formation of heteroduplex DNA
5. Removal of Holliday junctions by
DNAse cutting
6. The type of cutting affects whether
recombinants are seen or not
3. Migration of Holliday
junction
DS break repair model
(more likely)
Differences:
1. Both strands of
1 molecule break.
2. Strand digestion
and strand extension
occur.
3. Junctions do not slide.
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How does all this information get out of the DNA into the cell?
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