I.
Chapter 12
DNA Replication and Recombination
Fig 12.1
Three possible modes of replication
I.
D.
1.
2.
3.
Fig 12.2
DNA replication
Three possible modes of replication
A. Conservative—entire original molecule
maintained
B. Semiconservative—one strand is template for
new one
C. Dispersive – strands are a mixture of old and
new
DNA replication
Meselson and Stahl (1958) experiment
All DNA labeled with 15N
Shift to 14N for one generation
Density gradient centrifugation
Fig 12.3
Evidence for semi-conservative replication
1
I.
DNA replication
Theta replication: E. coli and other
organisms with circular DNA
Three types of DNA replication
1. Theta
2. Rolling circle
3. Linear
Fig 12.4
First a brief overview of the differences between
these, then the common details.
Linear replication: eukaryotic
chromsomes
Fig 12.5
Rolling circle replication: some
viruses and the F factor
Fig 12.6
2
The mantra: DNA synthesis occurs
from 5’ to 3’ on a 3’ to 5’ template.
DNA synthesis occurs by addition of
deoxyribonucleotide triphosphates (dNTPs)
Fig 12.7
Fig 12.8
DNA synthesis
occurs continuously
on the leading strand
and discontinuously
on the lagging strand
Fig 12.1
Fig 12.10
Fig 12.10
Fig 12.10
3
Initiation:
Unwinding:
• Inititator proteins
• Helicase
• Single-stranded
binding proteins
• Helicase: binds to the lagging strand template at each fork
and moves in a 5’ to 3’ direction
• Gyrase: relieves the strain ahead of the replication fork
Fig 12.12
Fig 12.11
Priming (primase)
Elongation (DNA polymerase III)
Fig 12.13
Fig 12.14
Primer replacement
(DNA polymerase I)
Backbone repair (ligase)
Fidelity of DNA replication
i) Nucleotide selection
ii) Proofreading
iii) Mismatch repair (Chapter 17)
Replication differs at the end
Fig 12.19
Fig 12.16
4
Summary
1. Replication is always semiconservative
2. Replication always begins at ‘origins’
The mantra: DNA synthesis occurs
from 5’ to 3’ on a 3’ to 5’ template.
3. DNA synthesis requires a primer
4. Elongation is always 5’ to 3’ on a 3’ to 5’
template
5. New DNA is synthesized from dNTPs
Summary
6. Replication is continuous on the leading
strand and discontinuous on the lagging
strand
7. New strands are complementary and
antiparallel to their template
8. Replication is fast and accurate due to
precise nucleotide selection,
proofreading, and repair
• Inititator proteins: bind to origin of replication and
cause DNA to unwind
• Single-stranded binding proteins
• Helicase: binds to the lagging strand template at each
fork and moves in a 5’ to 3’ direction; break H-bonds
• Gyrase: (topoisomerase) relieves the strain ahead of
the replication fork; dsDNA break and resealing
• Primase: lays down the RNA primer
• DNA polymerase III: replication of DNA beginning at
primer and extending from 5’ to 3’
• DNA polymerase I: primer replacement
• Ligase: backbone closure
The end replication problem
Eukaryotic DNA polymerase
•
•
•
•
There are at least 13! (Table 12.5)
DNA pol α: primase activity
DNA pol δ: replication on leading and
lagging strand
DNA pol β: recombination and repair
DNA pol γ: mtDNA replication
Fig 12.19
5
Fig 12.20
ssDNA end is
G-rich repeat
RNA of telomerase
is complementary
Telomerase synthesizes
complementary DNA
RNA template moves
Etc……
II.
A.
Recombination
Involves breakage/reunion of both strands
Synthesis on complementary
strand
Fig 12.22
Fig 12.22
If crossing over took place before
DNA synthesis it would look like
this:
…but crossing over results in
recombinant and nonrecombinant
products. Therefore it must take
place after DNA synthesis
6
Fig 12.23
V.
B.
Recombination
Holliday model: single stranded break
model
1.
Break both strands of each duplex
Note spelling: Holliday, not holiday.
Fig 12.23
Fig 12.23
2.
Broken strands pair with complement in
other duplex
4. Strand migration
3. Ligation
Fig 12.23
Fig 12.23
4. Resolution of the complex
7
Fig 12.23
Fig 12.23
Two possible types of resolution:
Fig 12.23
C.
1.
2.
3.
4.
C.
5.
6.
7.
8.
Double-stranded break model
Double strand break in one chromosome
Removal of nucleotides on one strand
3’ end displaces strand on unbroken
chromosome
DNA synthesis from the 3’ end
Double-stranded break model
Displaces strand pairs with broken end
DNA synthesis from the 3’ end of
nonmigrating strand
Ligation to form two Holliday junctions
Resolution of the junctions
8
D.
1.
2.
3.
4.
5.
6.
7.
8.
Enzymes involved in recombination
RecA – single strand invasion
RecBCD – unwinds double-stranded DNA
RuvA, RuvB – branch migration
Resolvase (RuvC) – cleaves Holliday structures
Single-stranded binding proteins
Ligase
DNA polymerases
gyrase
9