DNA: REPLICATION,
RECOMBINATION AND REPAIR
Objectives
• The mode of DNA synthesis
 The Meselson-Stahl experiment
 The semiconservative replication
 Replication forks and origins
• Synthesis of DNA
 DNA polymerases I,II,III
 Fidelity of synthesis (self study)
• DNA synthesis; A model
 Unwinding of the helix
 Initiation of synthesis
 Continuous and discontinuous synthesis
• DNA repair
Objectives
• Prokaryotic and Eukaryotic DNA synthesis
• DNA recombination
DNA replication
• In a cell, DNA replication begins
at specific locations in the
genome, called "origins".
• Unwinding of DNA at the origin,
and synthesis of new strands,
forms a replication fork.
• In addition to DNA polymerase,
the enzyme that synthesizes the
new DNA by adding nucleotides
matched to the template strand,
a number of other proteins are
associated with the fork and
assist in the initiation and
continuation of DNA synthesis.
• Cellular proofreading that ensure
near perfect fidelity for DNA
replication.
The Central Dogma
• The Flow of
Information: DNA →
RNA → Protein
• A gene is expressed in
two steps: DNA is
transcribed to RNA.
Then RNA is translated
into protein
DNA replication
• DNA replication, the basis for biological inheritance, is
a fundamental process occurring in all living organisms
to copy their DNA.
• In the process of "replication" each strand of the
original double-stranded DNA molecule serves as
template for the reproduction of the complementary
strand.
• Two identical DNA molecules have been produced from
a single double-stranded DNA molecule.
DNA Replication
• DNA replication is semiconservative (Meselson and Stahl
(1958))
– One strand goes to the next generation
– The other is new
• Each strand is a template for the other
– If one strand is 5’ AGCT 3’
– The other is
3’ TCGA 5’
DNA REPLICATION
• Write the strand complementary to
– 3’ ACTAGCCTAAGTCG 5’
– Answer
• DNA occurs in many forms-linear,circular,single stranded or
double stranded.
• DNA initiates at specific origins
– Unique origin in bacteria
– Multiple origins in eukaryotic chromosomes
• Is catalysed by DNA polymerases
– E coli – DNA polymerase I and DNA polymerase III
– Eukaryotes- α, ε and δ
DNA REPLICATION
• Challenges faced by the cell during replication
– How to separate two DNA strands- the cell has to protect the
unwound DNA from the action of nucleases that actually attack single
strands of DNA.
– Synthesis of DNA from the 5’ to the 3’ end- two strands have to be
synthesized in the same direction on anti-parallel templates (meansthe template has 5’-3’ strand and one 3’- 5’ strand as does the new
synthesized DNA.
– How to guard against errors in replication (How do we ensure that the
correct base is added to the growing polynucleotide chain.
Three possible models were proposed for DNA replication:
a. Conservative model proposed both strands of one copy would
be entirely old DNA, while the other copy would have both
strands of new DNA.
b. Dispersive model was that dsDNA might fragment, replicate
dsDNA, and then reassemble, creating a mosaic of old and new
dsDNA regions in each new chromosome.
c. Semiconservative model is that DNA strands separate, and a
complementary strand is synthesized for each, so that sibling
chromatids have one old and one new strand. This model was
the winner in the Meselson and Stahl experiment.
Three models for the replication of DNA
Peter J. Russl, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
DNA Replication
• Is semiconservative
– Meselson and Stahl (1958)
The Meselson-Stahl Experiment
The Meselson-Stahl Experiment
1. Meselson and Stahl (1958) grew E. coli in a heavy (not
radioactive) isotope of nitrogen, 15N in the form of
15NH Cl. Because it is heavier, DNA containing 15N is more
4
dense than DNA with normal 14N, and so can be
separated by CsCl density gradient centrifugation.
2. Once the E. coli were labeled with heavy 15N, the
researchers shifted the cells to medium containing
normal 14N, and took samples at time points. DNA was
extracted from each sample and analyzed in CsCl density
gradients.
The Meselson-Stahl experiment, which showed that DNA replicates semiconservatively
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Equilibrium centrifugation of DNA of different densities in a cesium chloride
density gradient
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
3. After one replication cycle in normal 14N medium, all DNA had density
intermediate between heavy and normal. After two replication cycles,
there were two bands in the density gradient, one at the intermediate
position, and one at the position for DNA containing entirely 14N.
4. Results compared with the three proposed models:
a. Does not fit conservative model, because after one generation there is
a single intermediate band, rather than one with entirely 15N DNA and
another with entirely 14N DNA.
b. The dispersive model predicted that a single band of DNA of
intermediate density would be present in each generation, gradually
becoming less dense as increasing amounts of 14N were incorporated
with each round of replication. Instead, Meselson and Stahl observed
two bands of DNA, with the intermediate form decreasing over time.
c. The semiconservative model fits the data very well.
How is DNA replicated?
Replication Origins and forks
(Bidirectional replication)
• The DNA double helix unwinds at a specific point
known as the origin of replication (OriC).
• DNA replication proceeds toward the direction of
the replication fork (leading strand) on one strand
and away from the fork (lagging strand) on the
other strand or in one direction only.
• In eukaryotes, when 2 replication forks are too near, a
replication bubble forms
• The OriC consists of 245 base pairs-characterised
by the presence of repeated sequences of 9 and
13 bases(9mers and 13 mers).
Replication origins and forks
• Replication begins when proteins bind at a
specific site on the DNA known as the origin of
replication (ori).
• What is a replicon? (self study)
– Eukaryotic replication is similar to prokaryotic
replication but more complex? (why?)
• The closed circular DNA of prokaryotes usually
only has one origin of replication (ori)
– Linear eukaryotic DNA has multiple ori’s
Replication Origins and forks
Eukaryotes
Prokaryotes
What are the functions of DNA
polymerases?
DNA Polymerases, the DNA Replicating Enzymes
1. First isolation of an enzyme involved in DNA
replication was in 1957. Arthur Kornberg won
the 1959 Nobel Prize for this work. (DNA
polymerase I)
2. Additional DNA polymerases have been
isolated, including DNA polymerase II (1970),
DNA polymerase III (1971), DNA polymerase
IV, and DNA polymerase V.
Properties of three bacterial DNA
polymerases ( in vivo)
• POL I,II III- all possess a 3’ to 5’ exonuclease activity- they can polymerize
in one direction, and then reverse directions and excise nucleotides just
added. The activity provides a capacity to proofread and remove incorrect
nucleotides. Only POL I can initiate DNA synthesis on a template
• Pol I/(III)- possess the 5’ to 3’ exonuclease activity. The activity allows the
enzyme to excise nucleotides from the end where initial synthesis
occurred and in the same direction. They have a potential ability to
remove the primer, an important aspect of synthesis. POL II and III can
elongate an existing strand (primer- RNA)
Roles of DNA Polymerases in vivo
• POL I- responsible for removing the primer as well as the
synthesis that fills in the gaps. Its exonuclease activity allows
for proofreading. It requires a primer and a DNA template to
assist in DNA synthesis.
• POL II – suspected to be involved in repair synthesis of DNA
that has been damaged by external forces like ultraviolet light.
• POL III- It is responsible for the polymerization essential to
replication. Its 3’ to 5’ exonuclease activity allows it to proof
read,excise and then correct base pairs created in error during
polymerization. Its active form is known as a holoenzyme.
What constitutes a holoenzyme (POL III)?
Roles of DNA Polymerases
1. All DNA polymerases link dNTPs into DNA chains . Main features of the
reaction:
a. An incoming nucleotide is attached by its 5’-phosphate group to the 3’OH of the growing DNA chain. Energy comes from the dNTP releasing
two phosphates. The DNA chain acts as a primer for the reaction.
b. The incoming nucleotide is selected by its ability to hydrogen bond
with the complementary base in the template strand. The process is
fast and accurate.
c. DNA polymerases synthesize only from 5’ to 3’.
2. The enzyme Kornberg isolated was believed to be the only DNA
polymerase in E. coli. The were two requirements for the in vitro DNA
synthesis
 All four deoxyribonucleoside triphosphates (dATP,dCTP,dGTP, dTTP = dNTP)
 Template DNA
DNA chain elongation catalyzed by DNA polymerase
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fidelity of synthesis (self study)
•
•
•
•
Kornberg (isolated DNA polymerase I).
Objective of his study
What was his approach?
Briefly describe the method used to test
fidelity of synthesis (nearest-neighbour
frequency test).
How is DNA replicated in
prokaryotic cells?
objectives
- Construct a model of DNA to stimulate DNA
replication.
- Understand why are RNA primers used in DNA
replication
- Understand how the DNA helix unwinds, initiates
synthesis (primer introduction), continuous and
discontinuous.
- Link the process of DNA replication to the
behavior of chromosomes in mitosis and meiosis
(cell cycle)
- Repair mechanisms (self study)
Molecular Model of DNA Replication
1.Table:1 shows key genes and DNA sequences
involved in replication.
DNA Synthesis
• DNA synthesis: A model
– A mechanism must exist by which the helix undergoes localised
unwinding and is stabilised in the open configuration so that the
synthesis may proceed along both strands.
– As unwinding proceeds, increased coiling creates tension further down
the helix which must be reduced.
– A primer must be synthesized so that polymerization can commence
under the direction of polymeraseIII
– When polymerase III commences synthesis of the compliment of both
strands of the parent molecule, the two strands are anti-parallel to
another. The other becomes continuous and the other becomes
discontinuous in the opposite direction.
– The primers must be removed before the completion of replication.
Gaps that are created must be filled with DNA complementary to the
template at each location.
– The newly synthesized DNA strand that fills each gap must be ligated
to the adjacent strand of DNA
UNWINDING OF DNA
Unwinding of DNA
-The OriC consists of 245 base pairs-characterised by the presence of
repeated sequences of 9 and 13 bases(9mers and 13 mers).
– - DNA helicase: unwinds the double helix by breaking the Hbonds at the replication fork(region where enzymes replicating
DNA bind to an untwisted, s.s. DNA strand). Helicases require energy
supplied by the hydrolysis of ATP in order to break hydrogen bonds and
denature the double helix. dnaA is responsible for the initial step in
unwinding the helix. A number of its subunits bind to each of the several
9mers. It is essential for subsequent binding of dnaB and dnaC proteins
that further open and destabilise the helix.
• SSBPs- The single stranded binding proteins (SSBs) bind to the exposed
bases to prevent them from annealing . Stabilises the conformation.
• DNA gyrase: relieves tension from the unwinding of the DNA strands
during bacterial replication. It cuts nicks in both strands of DNA, allowing
them to swivel around one another and then resealing the cut strands
Initiation, continuous and
discontinuous DNA synthesis
• In prokaryotes, there are 3 enzymes known to
function in replication & repair
– DNA polymerase I, II & III
• In eukaryotes,
there are 7 enzymes
known to function
in replication & repair
Building Complimentary Strands in
prokaryotes
• RNA primers are synthesized by primase and
are temporary
• The leading strand (uses 3’-5’ template) is
synthesized continuously
• The lagging strand (uses 5’-3’ template) is
synthesized discontinuously in short
fragments
Building Complimentary Strands in
prokaryotes
• DNA polymerase III builds the complimentary
strand of DNA
• DNA polymerase III adds complimentary
nucleotides (deoxyribonucleoside
triphosphates) in the 5’ to 3’ direction, using
RNA primers as starting points
– The segments are called Okazaki fragments
Building Complimentary Strands in
prokaryotes
• DNA polymerase I removes the RNA primers
from the leading strand and fragments from
the lagging strand and replaces them with the
appropriate deoxyribonucleotides.
Building Complimentary Strands in
prokaryotes
• DNA ligase joins the Okazaki fragments into one
strand on the lagging strand of DNA through the
formation of a phosphodiester bond.
Building Complimentary Strands in
prokaryotes
• As the 2 new strands of DNA are synthesized, 2
d.s. DNA molecules are produced that
automatically twist into a helix.
Initiation, continous and discontinous
synthesis
Summary of DNA synthesis
• Synthesis is initiated at a specific origin within each replicon.
In E.coli is called OriC
• Unwinding proteins (helicases) denature the DNA helix at the
origin, while the SSBPs stabilize the denatured DNA
• Synthesis is bidirectinal, creating two replication forks which
move in opposite directions away from the origin
• As the replication fork moves away from the origin, increased
coiling of the helix occurs and DNA gyrases diminishes the
tension. It cuts and seals DNA strands after uncoiling occurs.
• Initiation of DNA synthesis involves a RNA primer synthesized
under the direction of a primase. The resultant RNA is
complementary to its DNA template
Summary of DNA synthesis
• DNA polymerase III polymerizes complementary DNA strands
by elongating an existing chain in the 5’ to 3’ direction.
• As the replication forks move away from the origin, synthesis
is continuous on the leading strand, but is discontinuous on
the lagging strand, producing short strands known as okazaki
fragments.
• The RNA primers are removed and the resulting gaps are filled
with DNA under the direction of DNA polymerase I
• Along the lagging strand, the Okazaki fragments are joined by
DNA ligase.
Summary of DNA synthesis
• As this process proceeds along the length of
the replicon, proofreading by DNA polymerase
I and III occurs and semi conservative
replication is achieved
DNA Repair
DNA Repair (Proofreading)
• DNA polymerase III and DNA polymerase I
proofread the newly synthesized DNA strands.
• When mistakes occur, either enzyme can
function as an exonuclease.
– The enzyme backtracks and excises the incorrectly
paired nucleotide
– Then it continues forward adding nucleotides to
the complimentary strand
DNA Repair
• Repairs must be made immediately to avoid
errors being copied in subsequent
replications.
• Errors missed by proofreading can be
corrected by one of several repair mechanisms
that operate after the completion of DNA
replication.
Self study
• DNA damage repair mechanisms
– Excision repair
– Mismatch repair
– The SOS response
– Double strand Break repair
Replication of circular DNA and the supercoiling
problem
1. Some circular chromosomes (e.g., E. coli) are
circular throughout replication, creating a
theta-like (θ) shape. As the strands separate
on one side of the circle, positive supercoils
form elsewhere in the molecule. Replication
fork moves about 500 nt/ second, so at 10
bp/turn, replication fork rotates at 3,000 rpm.
2. Topoisomerases relieve the supercoils,
allowing the DNA strands to continue
separating as the replication forks advance.
Rolling Circle Replication
1. Another model for replication is rolling circle, which is
used by several bacteriophages, including ΦX174 (after a
complement is made for the genomic ssDNA) and λ (after
circularization by base pairing between the “sticky”
ssDNA cos ends)
2. Rolling circle replication begins with a nick (singlestranded break) at the origin of replication. The 5’ end is
displaced from the strand, and the 3’ end acts as a
primer for DNA polymerase III, which synthesizes a
continuous strand using the intact DNA molecule as a
template.
3. The 5’ end continues to be displaced as the circle “rolls”,
and is protected by SSBs until discontinuous DNA
synthesis makes it a dsDNA again.
The replication process of double-stranded circular DNA molecules through
the rolling circle mechanism
Chapter 3 slide 56
Prokaryotic DNA replication in
bacteriophages
Bacteriophage M13
•
On infecting an E.coli cell, the viral strand
directs the synthesis of its complemetary strand
to form a circular duplex replicative form (RF),
which can either be nicked (RF II) or supercoiled
(RF I).
•
As the M13(+) strand enters the E.coli cell, it
becomes coated by SSB except a segment of 57nt that form the hairpin.
RNA polymerase commences primer synthesis
6nt before start of the hairpin and extends the
RNA 20 to 30 residues to form a segment of
RNA-DNA hybrid duplex.
The DNA is displaced from the hairpin and
becomes coated with SSB so that when RNA
polymerases reaches it, primer synthesis stops.
Pol III holoenzyme then extends the RNA primer
around the circle to form the (-) strand.
The primer is removed by Pol I catalyzed nicktranslation, thereby forming RFII, which is
converted to RF I by sequential actions of DNA
ligase and DNA gyrase.
•
•
•
•
Prokaryotic DNA replication in
bacteriophages (Self study)
Bacteriophage φX174
•
The reaction sequence begin as that of M13. the
(+) strand is coated with SSb except for a 44 unit
hairpin. A 70-nt sequence containing hairpin is
known as pas (primosome binding site), which is
recognised & bound by the PriA, Pri B and Pri C
proteins.
•
DNaB and DNaC proteins are added to the DNA
with help of DNaT protein in an ATP requiring
process. DNaC is then released yielding a
preprimosome, which in turn binds primase
yielding the primasome.
•
At randomly selected sites, the primasome
reverses its migration while primase synthesizes
an RNA primer..
•
Pol III holoenzyme extends the primers to form
Okazaki fragments.
•
Pol I excises the primers and then replaces them
by DNA. The fragments are then joined by DNA
ligase and supercoiled by DNA gyrase to form
φX174 RF I.
How is DNA replicated in
eukaryotic cells?
DNA Replication in Eukaryotes
1.DNA replication is very similar in both
prokaryotes and eukaryotes, except that
eukaryotes have more than one chromosome.
Replicons
1. Eukaryotic chromosomes generally contain much
more DNA than those of prokaryotes, and their
replication forks move much more slowly. If they
were like typical prokaryotes, with only one origin
of replication per chromosome, DNA replication
would take many days.
2. Instead, eukaryotic chromosomes contain multiple
origins, at which DNA denatures and replication
then proceeds bidirectionally until an adjacent
replication fork is encountered. The DNA replicated
from a single origin is called a replicon, or
replication unit
3. In eukaryotes, replicon size is smaller than it is in
prokaryotes, replication is slower, and each
chromosome contains many replicons. Number and
size of replicons vary with cell type.
4. Not all origins within a genome initiate DNA
synthesis simultaneously. Cell-specific patterns of
origin activation are observed, so that
chromosomal regions are replicated in a
predictable order in each cell cycle
Fig. 3.13 Temporal ordering of DNA replication initiation events in replication units
of eukaryotic chromosomes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Eukaryotic Replication Enzymes
1. Enzymes of eukaryotic DNA replication aren’t as well
characterized as their prokaryotic counterparts. The
replication process is similar in both groups—DNA
denatures, replication is semiconservative and
semidiscontinuous and primers are required.
2. Fifteen DNA polymerases are known in mammalian cells:
a. Three DNA polymerases are used to replicate nuclear DNA. Pol α
(alpha) extends the 10-nt RNA primer by about 30nt. Pol δ(delta)
and Pol ε(epsilon) extend the RNA/DNA primers, one on the
leading strand and the other on the lagging (it is not clear which
synthesizes which).
b. Other DNA pols replicate mitochondrial or chloroplast DNA, or are
used in DNA repair.
Eukaryotic DNA Polymerases
• –DNA polymerases in
eukaryotes
• At least 7 polymerases: α, β,
γ, δ, ε, ς, η
• Polymerases α, δ and ε work
together in the replication
of nuclear DNA
• Polymerases β, ς, η and ε
function in nuclear DNA
repair
• Polymerase γ functions in
the replicationof
mitochondrial DNA
• DNA replication
• Properties:
– Replication is semiconservative
– Replication is directional: 5’ -> 3’
– Replication requires:
• A template
• A primer
Initiation of Replication (SELF STUDY)
1. Eukaryotic origins are generally not well characterized; those of the
yeast Saccharomyces cerevisiae are among the best understood.
2. Chromosomal DNA fragments (about 100bp) that are able to replicate
autonomously when introduced into yeast as extracellular, circular
DNA are known as ARSs (autonomously replicating sequences).
3. ARSs are yeast replicators. The three sequence elements typically
found in ARSs are A, B1, and B2.
4. Initiator protein in yeasts is the multiprotein origin recognition
complex (ORC), which binds to A and B1. Other replication proteins
join, including one that unwinds DNA at B2. The yeast origin of
replication is between regions B1 and B2.
5. DNA and histones must be doubled in each cell cycle. G1 prepares the
cell for DNA replication, chromosome duplication occurs during S
phase, G2 prepares for cell division, and segregation of progeny
chromosomes occurs during M phase, allowing the cell to divide.
6. Cell cycle control is complex, and only outlined here.
Yeasts, in which chromosomal replication is well studied,
serve as a eukaryotic model organism.
7. Initiation of replication has two separate steps,
controlled by cyclin-dependent kinases (Cdks) that are
present throughout the cell cycle, except during G1.
a. In the absence of Cdks during G1, replicator selection occurs.
ORC and other proteins assemble on each replicator to form
pre-replicative complexes (pre-RC).
b. When cell enters S phase, Cdks are present, and activate preRCs to initiate replication.
c. Cdk activity inhibits another round of pre-RC formation until the
cell again enters G1, when Cdks are absent.
How are ends of chromosomes
Replicated?
Replicating the Ends of Chromosomes
1. When the ends of chromosomes are
replicated and the primers are removed from
the 5’ ends, there is no adjacent DNA strand
to serve as a primer, and so a single-stranded
region is left at the 5’ end of the new strand. If
the gap is not addressed, chromosomes would
become shorter with each round of replication
The problem of replicating completely a linear chromosome in eukaryotes
Chapter 3 slide 72
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
2. Most eukaryotic chromosomes have short, species-specific sequences
tandemly repeated at their telomeres. Blackburn and Greider have
shown that chromosome lengths are maintained by telomerase, which
adds telomere repeats without using the cell’s regular replication
machinery.
3. In the ciliate Tetrahymena, the telomere repeat sequence is 5’
TTGGGG-3’
a.Telomerase, an enzyme containing both protein and RNA, binds to the
terminal telomere repeat when it is single stranded, synthesizing a 3-nt
sequence, TTG.
b.The 3’ end of the telomerase RNA contains the sequence AAC, which
binds the TTG positioning telomerase to complete its synthesis of the
TTGGGG telomere repeat.
c.Additional rounds of telomerase activity lengthen the chromosome by
adding telomere repeats.
4. After telomerase adds telomere sequences, chromosomal replication
proceeds in the usual way. Any shortening of the chromosome ends is
compensated by the addition of the telomere repeats.
5. If the sequence of the telomerase RNA is mutated, telomeres will
correspond to the mutant sequence, rather than the organism’s
normal telomere sequence. Using an RNA template to make DNA,
telomerase functions as a reverse transcriptase called TERT
(telomerase reverse transcriptase).
6. Telomere length may vary, but organisms and cell types have
characteristic telomere lengths. Mutants affecting telomere length
have been identified, and data indicate that telomere length is
genetically controlled. Shortening of telomeres eventually leads to cell
death, and this may be a factor in the regulation of normal cell death.
Synthesis of telomeric DNA by telomerase
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
How is the Genetic Information
rearranged by Genetic
recombination?
THEORETICAL PROBLEM AND SOLUTION
Homologous Recombination
• I. Overview of homologous recombination (the breakage and
rejoining of DNA into new combinations at sites of
significant sequence similarity)
• A. Requirement 1: identical or very similar sequences in the
crossover region: homologous recombination does not occur
between dissimilar sequences
• B. Requirement 2: complementary base pairing between
double-stranded DNA molecules
Homologous Recombination
• C. Requirement 3: heteroduplex formation: if similar
complementary sequences base pair, mismatches must exist,
unless the sequences are identical
• D. Requirement 4: recombination enzymes must catalyze the
various covalent and noncovalent rearrangement.
The Holliday model for genetic
recombination
•
•
•
•
Figure 1-1. The Holliday model for
genetic recombination.
One strand of each DNA molecule is
cut at the same position and then
pairs with the other molecule to
form a heteroduplex (region of blue
paired with black).
The strands are then ligated to form
the Holliday junction. This DNA
structure can isomerize between
forms I and II. Cutting and ligating
resolves the Holliday junction.
Depending on the conformation of
the junction, the flanking markers A,
B, a, and b will recombine or remain
in their original configuration.
The product DNA molecules will
contain heteroduplex patches.
Migration of Holliday junctions
• Figure 1-2. Migration of Holliday
junctions.
• By breaking the hydrogen bonds
holding the DNAs together in
front of the branch and reforming them behind, the
junction migrates and extends
the regions of pairing (i.e., the
heteroduplexes) between the
two DNAs.
• The heteroduplex region is
crosshatched. In the example,
two mismatches, GA and CT,
form in the heteroduplex region
because one of the DNA
molecules has a mutation in this
region.
Single-strand invasion model (self
study)
•
Figure 1-3. The single-strand invasion
model. A single-stranded break in
one of the two DNA molecules frees
a single-stranded end that invades
the other DNA molecule. The gap left
on the cut DNA is filled by DNA
polymerase (dashed line). The
displaced strand on the other DNA
molecule is degraded, and the two
ends are joined (arrows). Initially, a
heteroduplex, represented by
crosshatching, will form on only one
of the two DNA molecules. Branch
migration will cause another
heteroduplex to form on the other
DNA molecule. Isomerization can
recombine the flanking DNA
molecules, as in the Holliday model
Double-strand break repair model
•
Figure 1-4. The double-strand break
repair model. (1) A double-strand
break in one of the two DNAs
initiates the recombination event.
The arrows indicate the degradation
of the 5' ends at the break. (2) One
3' end, or tail, then invades the other
DNA, displacing one of the strands.
(3) This 3' end serves as a primer for
DNA polymerase, which extends the
tail until it can eventually be joined
to a 5' end (black arrow). Meanwhile, the displaced strand (in black)
serves as a template to fill the gap
left in the first DNA (dashed lines).
Two Holliday junctions form and
may produce recombinant flanking
DNA, depending upon how they are
resolved
Self study
• How are RNA genomes Replicated?
– Reverse transcriptase
• Enzymes of General Recombination
– RecA
– RecBCD
– RuvA,B and C