Chapter 25 DNA Metabolism
1,How does a DNA molecule replicate with
high fidelity?
2,How are DNA lesions (damages) repaired
to maintain the integrity of genetic
information?
3,How do DNA molecules recombine
(rearrange)?
High accuracy,multitude of participants.
1,The deduced double helix structure of
DNA revealed the possible ways for its
replication (1953)
? Each DNA strand was proposed to act as the
template (complement) of the other.
? The way a DNA molecule replicates was
hypothesized to be semiconservative,each of the
newly synthesized DNA duplexes consists of one
strand from the parent DNA and one strand of newly
synthesized (Watson and Crick,1953),(the
conservative replication would generate two
daughter DNA molecules with one consisting of two
new and one of two old strands.)
Old strandNew strand
The hypothesis of
semiconservative
replication proposed
by Watson and Crick
in 1953.
2,DNA replication was proved to be
semiconservative by the Meselson-Stahl
experiment using E,coli cells (1957)
? 15N (the Heavy isotope) and 14N (the Light isotope)
was used (as NH4Cl) to label the DNA to distinguish
the old and newly synthesized DNA molecules in
cells;
? Three types of DNA molecules containing various
proportions of 15N and 14N (H-H,H-L,L-L) were
separated by centrifugation to equilibrium in a
cesium chloride (CsCl) density gradient,
Radioisotope labeling
and density gradient
centrifugation clearly
distinguishes
replications of
semiconservative
from conservative.
The Meselson-
Stahl experiment:
DNA molecules
duplicate
semiconservatively
in E,coli cells.
15N-15N
0 generation
1 generation
2 generations
3 generations
4 generations
0 and 2 mixed
0 and 4 mixed
15N-14N 15N-15N14N-14N
BottomTop
3,A variety of simple questions were
asked about DNA replication
? Are the two parental strands completely unwound
before replication begins?
? Does replication begin at random sites or at unique
sites?
? Does DNA replication proceed in one direction or
both directions?
? The overall chain growth occurs in 5` 3`,3` 5`,or
both directions?
? What mechanisms ensure that DNA replicates once
per cell division?
? What enzymes take part in DNA synthesis?
? How does duplication of the long helical duplex
occur without the strands becoming tangled? …...
4,Autoradiography studies,daughter
strands are synthesized immediately
after parental strands separate
? Electron micrographs of the autoradiography of
replicating plasmid,SV40 virus,and E,coli
chromosomal DNA with 3H-thymidine incorporated
revealed ?-like structures,no single stranded DNA
was visible.
? The chromosomal DNA of E,coli is a single huge
circle!
? No temporary creation of linear DNA occurred
during replication of the circular DNA.
A electron micrograph of the replication
intermediate of a plasmid DNA,?-shaped
structures were observed; no single stranded
DNA is visible.
No complete unwinding of the two
parental strands occurred before the
daughter strands are synthesized
Replicating
SV40 DNA
Unreplicated,
positive supercoils
of parent strands
Replicated DNA
No complete unwinding of chains!
Autoradiogram of a
replicating E.coli
chromosomal DNA
labeled by [3H]thymidine.
No complete
unwinding of
DNA chains!
5,DNA replication was found to begin at
specific sites and proceed bidirectionally
? Pulse-chase labeling studies of replicating DNA,as
well as direct EM examination of intermediates of
replicating linear T7 bacteriophage DNA all
revealed that DNA replication is bidirectional.
? Denaturation mapping studies with a series of
replication intermediates of circular DNA and direct
observation of a those of linear DNA revealed that
DNA replication begins at specific replication
origins.
Autoradiogram of replicating mammalian
cellular DNA pulse-chase labeled with
[3H]thymine,replication is bidirectional.
Examination of T7 DNA (linear) replication using electron microscopy:
1,The daughter polynucleotide strands are synthesized almost as soon as
the parental strands separate (no complete unwinding of chains);
2,Replication always began at a specific internal site (not from the ends,
not random);
3,The replication proceeds in both directions (determined by measuring
the distance between the replication fork and the ends)
Replication
origin
Replication
forks
Denatured loops
(single-stranded)
Nondenatured DNA
(double-stranded)
Denaturation mapping:
the denatured loops are
reproducible and thus can be
used as points of reference
(DNA replication starts at
specific origins).
6,The chemistry of DNA polymerization
was revealed by in vitro studies using a
DNA polymerase purified from E,coli
? Arthur Kornberg purified DNA polymerase I,a
monomeric 103 kDa protein,from E,coli in 1955
and revealed the major features of the DNA
synthesis process.
? DNA polymerase I was found to catalyze DNA
polymerization in vitro in the presence of a single-
stranded DNA template,a preexisting primer with a
free 3`-OH group and the dNTPs.
? The fundamental reaction for DNA synthesis is a
nucleophilic attack by the 3`-OH group of the
growing strand on the 5`-a-phosphorus of a
incoming dNTP selected via base-pairing,the newly
synthesized DNA is always extended in the 5` to 3`
direction.
7,DNA polymerase I has 3` 5`,as well
as 5` 3` exonuclease activities
? The 3` 5` exonuclease activity was found to be
able to remove mismatched base pairs,thus to
proofread the newly incorporated nucleotides
(increasing the polymerization accuracy by 100 to
1000 fold).
? A ―sliding back‖ model was proposed for DNA
polymerase I to proofread mismatched base pairs.
? The enzyme can be cleaved into two parts by mild
protease treatment,the small fragment contains the 5’
to 3’ exonuclease activity and the large (called the
Klenow fragment) contains the rest two activities.
? The 5` to 3` exonuclease activity of DNA
polymerase I enable it to catalyze the nick
translation process,an RNA or DNA strand paired
to a DNA template is simultaneously degraded and
replaced; an activity used for both DNA repair and
the removal of RNA primers in DNA replication,
also for incorporating radioisotope-labeled dNTPs
into a DNA probe.
DNA polymease I is
proposed to slide back
to proofread a mismatched
base pair using its 3` to 5`
exonuclease activity.
DNA polymerase I has three enzymatic
activities in a single polypeptide chain,
which can be cleaved into two functional
parts by mild protease treatment.
Protease cleavage
The 3` to 5`
exonuclease
domain
The polymerase
domain
Structure of the
Klenow fragment
of DNA polymease I
The 5` to 3` exonuclease
activity of DNA
polymerase I allows the
enzyme to catalyze the
nick translation process
of a DNA or RNA
fragment base paired
with a template,
32P-dATP
Arthur Kornberg won
the 1959 Nobel Prize
in Medicine for his
discovery of the
mechanism in the
biological synthesis of
deoxyribonucleic acid
(before Watson and
Crick won theirs!)
8,The synthesis of the two daughter
DNA strands was found to be
semidiscontinuous
? At a replication fork,the overall elongation direction
for one daughter strand is 5` to 3` and 3` to 5` for the
other due to the antiparallel features of DNA
duplexes,
? DNA polymerase catalyzing 3` to 5` extension was
hypothesized but never identified.
? Reiji Okazaki discovered ( in the 1960s) that a
significant proportion of newly synthesized DNA
exists as small fragments!
? These so-called Okazaki fragments was found to
join together by DNA ligases to form one of the
daughter strands;
? Thus both daughter strands are synthesized in 5` to
3` direction.
? One daughter strand at the replication fork is
synthesized continuously and the other
discontinuously,called the leading and lagging
strands respectively.
Overall direction of progeny chain growth
at a replicating fork,one in 5’ 3’ and the
other in 3’ 5’ direction.
Both daughter strands at the replication fork are synthesized in
5’ 3’ direction,but one (the leading strand) is synthesized
continuously and the other (the lagging strand) discontinuously
(synthesized initially as Okazaki fragments).
The leading strand
The lagging strand
9,DNA polymerase I was found to be
not responsible for DNA replication in
E,coli cells
? The reaction velocity of this enzyme is too low to
account for the observed rates of fork movement.
? Its processivity—the average number of nucleotides
added before it dissociate from the template-- is too
low (about 50 nucleotides).
? E.coli cells having a defective DNA polymerase I
were found to be still viable,although sensitive to UV
light (1969).
? Two more DNA polymerases (II and III) were
revealed in E.coli cells in the early 1970s and two
more in 1999.
10,DNA polymerase III is responsible
for DNA replication in E.coli cells
? DNA polymerase III is a multimeric enzyme
containing at least 10 different subunits.
? The holoenzyme exists as an asymmetric dimer (one
is believed to be used for synthesizing the leading
and the other the lagging strand).
? The polymerization and proofreading activities are
located on separate subunits,a and e respectively
(the enzyme has no 5` to 3` exonuclease activity).
? The b subunits provide high processivity,with more
than 500,000 nucleotides added per binding.
? The rate of DNA synthesis is high,with 1000
nucleotides/second).
Sliding clamp
subunits
Catalytic
subunit
Catalytic
subunit
3’ 5’ exonuclease
subunits
Clamp
loader
Proposed
architecture of DNA
polymerase III
holoenzyme,
an asymmetric
dimer
No 5` to 3` exonuclease
activity
DNA duplex
(infered)
A clamping
b dimer (determined)
The two b subunits of
E,coli polymerase III
form a circular clamp
that may surround
DNA to increase its
processivity.
11,DNA polymerase III and many other
proteins are part of the replisome for
DNA replication in E.coli cells
? In vitro studies revealed about 20 proteins are
involved in DNA replication in E.coli cells.
? These include,
– the helicase (dnaB) for moving along the DNA
and separating the two DNA strands using energy
from ATP;
– the topoisomerases for relieving topological
(torsional) strains in the helical structure (positive
supercoils) generated during strand separation;
– the DNA-binding protein for binding and
stabilizing the single stranded DNA generated;
– the primase (dnaG) for generating a short RNA
primer;
– the DNA polymerase III for polymerizing and
proofreading the nucleotides according to the
templates;
– the DNA polymerase I (polA) for removing the
RNA primers and replacing by a DNA sequence;
– the DNA ligase for sealing nicks between the
Okazaki fragments.
12,A 245 bp fragment in the E.coli
chromosomal DNA,OriC,was identified
to be the replication origin
? It was identified by using origin-lacking plasmids.
? OriC contains a tandem array of three 13-mer with nearly
identical sequences and four 9-mer repeats,all of which
highly conserved among all bacterial replication origins.
? OriC contains 9 GATC (palindromic) sequences,which can
be methylated at the base ring of A,which is believed to be
important in having only one replication per cell division.
OriC,the replication origin of E,coli
Chromosome,contains three repeats
Of 13 bp ad four repeats of 9 bp.
GATC 5GATC
13,The molecular details of the
replication of E,coli chromosomal DNA
are the best understood by in vitro studies
? The whole process can be divided into three stages,
initiation,elongation,and termination.
? During the initiation stage,the DNA is open at the
OriC site and a prepriming complex is formed,with
the participation of at least nine proteins.
? During the elongation stage,short RNA primers are
synthesized by the primase,extended by the DNA
polymerase III,and ligated by the DNA ligase (for
the Okazaki fragments of the lagging strand).
? During the termination stage,the two replication
forks meet at the Ter sequences,the replisome
dissociates,and the two catenated chromosomes are
separated by DNA topoisomerase IV.
14,Multiple proteins participate at the
initiation stage of DNA replication of the
E,coli chromosome
? The replication process begins when a single
complex of about 20 DnaA protein molecules (with
ATP bound) binds to the four 9 bp repeats at OriC
that is supercoiled,
? Aided by HU (a histone-like protein),the bound
DnaA complex opens the two DNA strands at the
AT-rich 13-mer repeats.
? DnaB,the helicase,binds (aided by DnaC protein)
to the opened DNA strands as two hexamer clamps
and further opens (unwinds) the strands in both
directions,thus forming the prepriming complex.
The prepriming complex is
formed at the OriC,with the
participation of DnaA,HU,
DnaB,DnaC and other
proteins.
The prepriming
complex
15,The leading strand is synthesized
continuously but the lagging strand
discontinuosuly
? Further unwinding of the DNA duplex needs DNA
gyrase ( a topoisomerase II) to relieve the supercoils
ahead the replication forks and SSB for stabilizing
the unwound single strand DNAs.
? Kornberg discovered that the nascent DNA is
always covalently linked to a short stretch of RNA.
? The RNA primers were found to be 10-60
nucleotides in length.
? The primase (a RNA polymerase),recruited to the
open templates via DnaB,was found to be the
enzyme that catalyzes the synthesis of the RNA
primers.
? For the leading strand,only one primer is
synthesized,which is then elongated by DNA
polymerase III in a continuous way.
? On the lagging strand,DnaB (the helicase)
intermittently recruits DnaG (the DNA primase) to
form a complex called primosome,and repeatedly
making primers for the Okazaki fragments,
? Each Okazaki fragment is then synthesized on one
RNA primer by DNA polymerase III.
? The RNA primers are removed and replaced by a
DNA sequence in a reaction catalyzed by DNA
polymerase I,using its nick translation activity (5`
to 3` exonuclease + polymerase).
? The final Okazaki fragments are joined together by
the DNA ligase.
? DNA ligase catalyzes the formation of a
phosphodiester bond between a 3`-OH group and a
5` phosphate group of two DNA strands,where the
5` phosphate is first activated by being modified by
an AMP group coming from NAD+ (the bacterial
enzyme) or ATP (the virus and animal enzymes),
Only one RNA primer is needed for
synthesizing the leading strand.
(DnaB)
RNA primers are
repeatedly formed
by the primase on
the lagging strand.
DNA polymerase I replaces the RNA primers by
DNA sequences and DNA ligase seals the nicks
DNA ligase first transfers
an AMP moiety to the 5`
phosphate group (from
NAD+ or ATP) before
making a phosphodiester
bond.
16,The leading and lagging strands are
believed to be synthesized coordinately by
a single asymmetric DNA polymerase III
dimer
? The two polymerase III cores (having the ae?
subunits) are believed to be connected by two t
subunits.
? The coupling is believed to accomplish via looping
of the lagging strand template.
? The PolIII on the lagging strand intermittently
releases the b sliding clamp and the completed
Okazaki fragment before rebinding to a new b
subunit dimer loaded at a RNA primer and
synthesizing a new Okazaki fragment.
? Accumulating evidence seem to support that the Pol
III dimer complex is associated with the plasma
membrane and does not actually itself move; the
DNA moves through the fixed complex.
Sliding clamp
subunits
Catalytic
subunit
Catalytic
subunit
3’ 5’ exonuclease
subunits
Clamp
loader
Proposed
architecture of DNA
polymerase III
holoenzyme,
an asymmetric
dimer
No 5` to 3` exonuclease
activity
17,E.coli DNA replication ends at a
specific terminus region with multiple
copies of a 20 bp Ter sequence
? The Ter sequences are positioned in two clusters
with opposite orientations.
? The Tus protein can bind to the Ter sequences.
? At each round of DNA replication,only one Tus-Ter
complex seem to function to arrest a replication fork
from one direction,with the opposing replication
fork stopping when the two collide.
? The Ter sequences do not seem to be essential for
stopping replication.
? The two newly synthesized circular chromosomal
DNAs are topologically interlinked (catenated) and
is finally separated by the action of a Type II
topoisomerases,
18,Methylation of GATC sequences at
oriC is believed to control the replication
frequencies in E,coli cells
? Hemimethylated oriC was found unable to initiate
another round of DNA replication.
? It is believed that the hemimethylated GATC sequences
at OriC is sequestered in the plasma membrane
immediately after one round of DNA replication,thus
blocking another round of replication until the GATC
sequences are released and fully methylated by Dam
methylase.
? The delay of methylation is hypothesized to limit DNA
replication to occur once per cell division,
? The slow hydrolysis of ATP by DnaA protein was also
proposed to regulate replication initiation.
Hemimethylatd OriC
is not active for
initiating DNA
replication
Active
Inactive
19,DNA replication in eukaryotic cells
use essentially the same principles but
more complex in the details
? The best understood eukaryotic replication system is
that of SV40 and yeast.
? Formation of the RNA primers and the initial
incorporation of deoxynucleotides are believed to be
catalyzed by DNA polymerase a (which has no
proofreading activity).
? Later chain extension is believed to be catalyzed by
DNA polymerase d,which has proofreading activity
and is
? Specific sequences (~150 bp) that can function as
replication origins have only been identified in yeast
( is called autonomously replicating sequences,or
ARS) and SV40 virus.
? The rate of DNA synthesis in eukaryotic cells is
about one tenth of that of the E.coli cells.
? Replication in the eukaryotic genomes begin at
many replication origins.
? 9 purified proteins are needed to replicate SV40
virus DNA in vitro.
? T antigen--a multifunctional site-specific DNA
binding protein encoded by SV40 DNA,binds to the
origin (as DnaA) and melts the duplex DNA (as
DnaB);
? RPA– encoded by the host mammalian cells and binds
to the melted single-stranded DNA (as SSB).
? Pol a/primase-- synthesizes the RNA primers and a
stretch of DNA sequences,has no proofreading activity.
? Pol d– replaces Pol a/primase to further extend the
RNA-DNA strand,has proofreading activity,
? PCNA (proliferating cell nuclear antigen)-- a trimeric
ring-shaped protein that clamps Pol d onto the DNA
template.
? RFC (replication factor C)-- a clamp loader for PCNA,
Topoisomerases-- Relieves the torsional strain induced
by the growing replication fork.
? Ligases--joins the Okazaki fragments (which is much
shorter than those in E.coli cells),as well as the leading
strand.
Model of in vitro replication
of SV40 DNA by eukaryotic
enzymes:
1,T antigen (Tag) binds and
unwinds replication origin;
2,RPA (or RFA) binds to
single-stranded DNA;
3,Pol a-primase
synthesizes the primers
(RNA + DNA).
4,RFC loads PCNA to the
template.
5,PCNA displaces
Pola-primase and
functions as a
DNA clamp;
6,Pol d replaces
Pola-primase
and further
extends the DNA
strands.
20,Maintaining the integrity of the
genomic DNA is essential to all cells
? The DNA molecules can become damaged via a
variety of internal (e.g.,autonomous deamination of
bases) or external (e.g.,exposure to UV light and
chemical agents) processes.
? A diversity of repair systems have evolved,
mismatch repair,base-excision repair,nucleotide-
excision repair and direct repair are the common
types found.
? Many DNA repair systems are energetically
expensive (very inefficient comparing with other
metabolic pathways) and redundant (especially to
some common types of lesions).
? Unrepaired DNA damages can introduce mutations,
which can cause cancers in mammals (e.g.,
xeroderma pigmentosum,着色性干皮病,is caused
by a defect of the DNA repair systems).
? Most carcinogens are found to be strong mutagens,
the Ames Test has been used to examine whether a
chemical is a potential human carcinogen by testing
its mutagenicity on certain bacterial strains.
? Complementary synthesis (based on the duplex
DNA structure) after elimination of the damaged
nucleotides is a common principle for all the repair
systems,
Ames Test:
carcinogens are
tested for their
mutagenicity on
bacterial genomes.
Plating of
his- salmonella
typhimurium
High level of mutagen
Medium level of mutagen Low level of mutagen
No mutagen Back
mutations
21,The mismatch repair pathway acts to
increase the fidelity of DNA replication
? In vitro studies showed that the transient
undermethylation of GATC sequences in the newly
synthesized strand permits strand discrimination in
E.coli (not in eukaryotic cells),mismatches are
corrected according to information provided by the
parent strand.
? At least 12 proteins are involved in mismatch repair in
E,coli,among which the MutS-MutL complex acts to
recognize all the mismatches (except C-C) and MutH
acts to recognized GATC sequences and generates a
nick on the 5` side of the G in the (5`) GATC in the
newly synthesized (unmethylated) strand when the two
meet.
? Exonucleases (I or X cleaves from 3` to 5`direction;
VII or RecJ cleaves from 5` to 3` direction) remove
a segment of DNA from the newly synthesized
strand including the mispaired nucleotide.
? DNA pol III resynthesizes the removed segment.
? DNA ligase reseals the gap on the newly
synthesized DNA.
The Mut L,MutS,and MutH
proteins in E,coli recognize
the mismatched base pairs
and generate a nick at a
GATC sequence up to 1kb
away.
MutL links MutS (recognizing the
mismatch and MutH (recognizing
the GATC sequence)
MutS
A proposed model for methyl-directed
mismatch repair in E,coli cells.
Mismatch on the 3` side
of the cleavage site Mismatch on the 5` side of the cleavage site
22,Base-excision repair acts by
removing the damaged bases first
? The key enzymes for this class of repair include the
specific DNA glycosylases (e.g.,uracil,hypoxanthine,
3-methyladenine,7-methylguanine,and pyrimidine
dimers glycosylases) and the nonspecific AP
(apurinic or apyrimidinic) endonucleases.
? The DNA glycosylases remove specific DNA lesions
by cleaving the N-glycosyl bonds.
? AP endonucleases cleave the phosphodiester bond
near the AP site (either on the 5` or the 3` side of AP).
? DNA polymerase I replaces a segment of DNA
including the AP site and DNA ligase seals the gap.
The proposed model
for base-excision
repair using specific
glycosylases and
nonspecific AP
endonucleases.
23,Nucleotide-excision repair acts by
removing a short fragment of ssDNA
containing the lesion
? Usually works when the DNA lesion (e.g.,the
presence of cyclobutane pyrimidine dimers or 6-4
photoproducts) causes large distortion in the helical
structure and is probably the most important way for
DNA repair in cells.
? in E,coli,ABC exinuclease,a complex of three
proteins (UvrA,UvrB,and UvrC),scans the lesion
(A2B),generates two cuts (3` side by B and 5` side by
C) on the two sides of the damaged site.
? A 12 mer segment of DNA spanning the damaged
bases is released by the UvrD helicase.
? DNA Pol I fills the gap and DNA ligase seals it.
? The exinuclease in eukaryotic cells contains 16
polypeptides,with none homologous to that of the
E.coli enzyme complex.
? A 29 mer is released by the human exinuclease.
24,Direct repair acts by directly reverse
the base modifications without removing
a base or nucleotide
? Pyrimidine dimers can be directly converted to two free
monomeric pyrimines in a reaction catalyzed by DNA
Photolyases via free radical intermediates.
? The methyl group of O6-methylguanine (introduced by
alkylating agents) is directly transferred to a Cys
residue on O6-methylguanine-DNA
methyltransferase (not an enzyme in strict sense).
? The methylated O6-methylguanine-DNA
methyltransferase act as a transcription activator for its
own gene and a few other genes also encoding repair
enzymes.
A proposed action
mechanism for the
photolyases
Chromophores
Three unstable radicals
O6-methylguanine-DNA
methyltransferase
Cys-S CH3
O6 methylguanine on DNA is
directely repaired by removing
the methyl group by a Cys
residue of the methyltranseferase
A transcription
activator
25 Recombinational repair or error-
prone repair have to occur when the
complementary strand is also damaged
or absent
? These types of lesions include double-strand breaks,
double strand cross-links,both complementary
strand are damaged or one is damaged with the other
absent (as will occur when a replication fork
encounters an unrepaired lesiono r strand break).
? The lesions can be repaired by using information
from a separate,homologous chromosome via
recombinational DNA repair (discussed later).
? When the DNA strands are extensively damaged,the
SOS response and error-prone repair (or translesion
replication,being a desperate strategy) will occur.
? Some proteins may act to repair lesions (the ABC
exinuclease,RecA,Pol II),some to inhibit cell divisions
(e.g.,sulA),and others to act in translesion replication
(Rec A protein,SSB,DNA polymerase V made of
UmuC and the shortened UmuD or DNA polymerase IV
encoded by the dinB gene).
? Random nucleotides may be added in place of the
damaged ones during translesion replications,but the
detailed mechanism has not been revealed.
? The increased mutations may generate a few cells
(although almost all dead) that are better fit to survive
the catastrophic conditions (better species may thus
evolve).
When a replication fork passes the unrepaired lesions
situations will be generated where both complementary
strands of a duplex are unable to act as a template.
26,DNA rearranges in cells via
genetic recombination
? Blocks of genes from homologous chromosomes
(e.g.,between homologous paternal and maternal
chromosomes) were found to be exchanged by the
process of crossing over,or homologous
recombination during Drosophila meiosis (early
20th century).
? Homologous recombination was then found to occur
commonly in all types of organisms between two
DNA sequences sharing homology.
? Two more major types of DNA rearrangements were
later revealed.
? Site-specific recombination was found to occur
when the DNA of l phage integrates into the E.coli
genome,where the DNA exchange occurs only
between specific DNA sequences.
? Site-specific recombination was also found to occur
in rearranging certain genes in all types of
eukaryotic cells.
? Certain DNA elements,called transposons,were
found to be able to randomly transpose (mobilize,
hop) from one location to another on chromosomes,
thus called DNA transposition (speculated by
Barbara McClintock while studying maize genetics
in the 1950s),which was also found in bacteria and
animals many years later.
? All the strands of the recombining DNA molecules
have to be broken and rejoined with new strands for
all these recombinations.
? Novel DNA intermediates with unusual structures
(with three or four strands interwound) have to be
formed.
? Principles of the three kinds of recombinations have
been widely used to carry out artificial
manipulations of DNAs (or genetic engineering) in
all types of cells.
Homologous recombination is believed to occur
between the closely associated chromatids (in tetrads)
during the prophase of the first meiotic division of
germ-line cells to produce haploid gametes.
Current studies show that these observed
“crossover” sites may not promote
but inhibit DNA exchange.
EM examination of a chiasmata,no contact
between homologous DNA molecules!
1,How does a DNA molecule replicate with
high fidelity?
2,How are DNA lesions (damages) repaired
to maintain the integrity of genetic
information?
3,How do DNA molecules recombine
(rearrange)?
High accuracy,multitude of participants.
1,The deduced double helix structure of
DNA revealed the possible ways for its
replication (1953)
? Each DNA strand was proposed to act as the
template (complement) of the other.
? The way a DNA molecule replicates was
hypothesized to be semiconservative,each of the
newly synthesized DNA duplexes consists of one
strand from the parent DNA and one strand of newly
synthesized (Watson and Crick,1953),(the
conservative replication would generate two
daughter DNA molecules with one consisting of two
new and one of two old strands.)
Old strandNew strand
The hypothesis of
semiconservative
replication proposed
by Watson and Crick
in 1953.
2,DNA replication was proved to be
semiconservative by the Meselson-Stahl
experiment using E,coli cells (1957)
? 15N (the Heavy isotope) and 14N (the Light isotope)
was used (as NH4Cl) to label the DNA to distinguish
the old and newly synthesized DNA molecules in
cells;
? Three types of DNA molecules containing various
proportions of 15N and 14N (H-H,H-L,L-L) were
separated by centrifugation to equilibrium in a
cesium chloride (CsCl) density gradient,
Radioisotope labeling
and density gradient
centrifugation clearly
distinguishes
replications of
semiconservative
from conservative.
The Meselson-
Stahl experiment:
DNA molecules
duplicate
semiconservatively
in E,coli cells.
15N-15N
0 generation
1 generation
2 generations
3 generations
4 generations
0 and 2 mixed
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3,A variety of simple questions were
asked about DNA replication
? Are the two parental strands completely unwound
before replication begins?
? Does replication begin at random sites or at unique
sites?
? Does DNA replication proceed in one direction or
both directions?
? The overall chain growth occurs in 5` 3`,3` 5`,or
both directions?
? What mechanisms ensure that DNA replicates once
per cell division?
? What enzymes take part in DNA synthesis?
? How does duplication of the long helical duplex
occur without the strands becoming tangled? …...
4,Autoradiography studies,daughter
strands are synthesized immediately
after parental strands separate
? Electron micrographs of the autoradiography of
replicating plasmid,SV40 virus,and E,coli
chromosomal DNA with 3H-thymidine incorporated
revealed ?-like structures,no single stranded DNA
was visible.
? The chromosomal DNA of E,coli is a single huge
circle!
? No temporary creation of linear DNA occurred
during replication of the circular DNA.
A electron micrograph of the replication
intermediate of a plasmid DNA,?-shaped
structures were observed; no single stranded
DNA is visible.
No complete unwinding of the two
parental strands occurred before the
daughter strands are synthesized
Replicating
SV40 DNA
Unreplicated,
positive supercoils
of parent strands
Replicated DNA
No complete unwinding of chains!
Autoradiogram of a
replicating E.coli
chromosomal DNA
labeled by [3H]thymidine.
No complete
unwinding of
DNA chains!
5,DNA replication was found to begin at
specific sites and proceed bidirectionally
? Pulse-chase labeling studies of replicating DNA,as
well as direct EM examination of intermediates of
replicating linear T7 bacteriophage DNA all
revealed that DNA replication is bidirectional.
? Denaturation mapping studies with a series of
replication intermediates of circular DNA and direct
observation of a those of linear DNA revealed that
DNA replication begins at specific replication
origins.
Autoradiogram of replicating mammalian
cellular DNA pulse-chase labeled with
[3H]thymine,replication is bidirectional.
Examination of T7 DNA (linear) replication using electron microscopy:
1,The daughter polynucleotide strands are synthesized almost as soon as
the parental strands separate (no complete unwinding of chains);
2,Replication always began at a specific internal site (not from the ends,
not random);
3,The replication proceeds in both directions (determined by measuring
the distance between the replication fork and the ends)
Replication
origin
Replication
forks
Denatured loops
(single-stranded)
Nondenatured DNA
(double-stranded)
Denaturation mapping:
the denatured loops are
reproducible and thus can be
used as points of reference
(DNA replication starts at
specific origins).
6,The chemistry of DNA polymerization
was revealed by in vitro studies using a
DNA polymerase purified from E,coli
? Arthur Kornberg purified DNA polymerase I,a
monomeric 103 kDa protein,from E,coli in 1955
and revealed the major features of the DNA
synthesis process.
? DNA polymerase I was found to catalyze DNA
polymerization in vitro in the presence of a single-
stranded DNA template,a preexisting primer with a
free 3`-OH group and the dNTPs.
? The fundamental reaction for DNA synthesis is a
nucleophilic attack by the 3`-OH group of the
growing strand on the 5`-a-phosphorus of a
incoming dNTP selected via base-pairing,the newly
synthesized DNA is always extended in the 5` to 3`
direction.
7,DNA polymerase I has 3` 5`,as well
as 5` 3` exonuclease activities
? The 3` 5` exonuclease activity was found to be
able to remove mismatched base pairs,thus to
proofread the newly incorporated nucleotides
(increasing the polymerization accuracy by 100 to
1000 fold).
? A ―sliding back‖ model was proposed for DNA
polymerase I to proofread mismatched base pairs.
? The enzyme can be cleaved into two parts by mild
protease treatment,the small fragment contains the 5’
to 3’ exonuclease activity and the large (called the
Klenow fragment) contains the rest two activities.
? The 5` to 3` exonuclease activity of DNA
polymerase I enable it to catalyze the nick
translation process,an RNA or DNA strand paired
to a DNA template is simultaneously degraded and
replaced; an activity used for both DNA repair and
the removal of RNA primers in DNA replication,
also for incorporating radioisotope-labeled dNTPs
into a DNA probe.
DNA polymease I is
proposed to slide back
to proofread a mismatched
base pair using its 3` to 5`
exonuclease activity.
DNA polymerase I has three enzymatic
activities in a single polypeptide chain,
which can be cleaved into two functional
parts by mild protease treatment.
Protease cleavage
The 3` to 5`
exonuclease
domain
The polymerase
domain
Structure of the
Klenow fragment
of DNA polymease I
The 5` to 3` exonuclease
activity of DNA
polymerase I allows the
enzyme to catalyze the
nick translation process
of a DNA or RNA
fragment base paired
with a template,
32P-dATP
Arthur Kornberg won
the 1959 Nobel Prize
in Medicine for his
discovery of the
mechanism in the
biological synthesis of
deoxyribonucleic acid
(before Watson and
Crick won theirs!)
8,The synthesis of the two daughter
DNA strands was found to be
semidiscontinuous
? At a replication fork,the overall elongation direction
for one daughter strand is 5` to 3` and 3` to 5` for the
other due to the antiparallel features of DNA
duplexes,
? DNA polymerase catalyzing 3` to 5` extension was
hypothesized but never identified.
? Reiji Okazaki discovered ( in the 1960s) that a
significant proportion of newly synthesized DNA
exists as small fragments!
? These so-called Okazaki fragments was found to
join together by DNA ligases to form one of the
daughter strands;
? Thus both daughter strands are synthesized in 5` to
3` direction.
? One daughter strand at the replication fork is
synthesized continuously and the other
discontinuously,called the leading and lagging
strands respectively.
Overall direction of progeny chain growth
at a replicating fork,one in 5’ 3’ and the
other in 3’ 5’ direction.
Both daughter strands at the replication fork are synthesized in
5’ 3’ direction,but one (the leading strand) is synthesized
continuously and the other (the lagging strand) discontinuously
(synthesized initially as Okazaki fragments).
The leading strand
The lagging strand
9,DNA polymerase I was found to be
not responsible for DNA replication in
E,coli cells
? The reaction velocity of this enzyme is too low to
account for the observed rates of fork movement.
? Its processivity—the average number of nucleotides
added before it dissociate from the template-- is too
low (about 50 nucleotides).
? E.coli cells having a defective DNA polymerase I
were found to be still viable,although sensitive to UV
light (1969).
? Two more DNA polymerases (II and III) were
revealed in E.coli cells in the early 1970s and two
more in 1999.
10,DNA polymerase III is responsible
for DNA replication in E.coli cells
? DNA polymerase III is a multimeric enzyme
containing at least 10 different subunits.
? The holoenzyme exists as an asymmetric dimer (one
is believed to be used for synthesizing the leading
and the other the lagging strand).
? The polymerization and proofreading activities are
located on separate subunits,a and e respectively
(the enzyme has no 5` to 3` exonuclease activity).
? The b subunits provide high processivity,with more
than 500,000 nucleotides added per binding.
? The rate of DNA synthesis is high,with 1000
nucleotides/second).
Sliding clamp
subunits
Catalytic
subunit
Catalytic
subunit
3’ 5’ exonuclease
subunits
Clamp
loader
Proposed
architecture of DNA
polymerase III
holoenzyme,
an asymmetric
dimer
No 5` to 3` exonuclease
activity
DNA duplex
(infered)
A clamping
b dimer (determined)
The two b subunits of
E,coli polymerase III
form a circular clamp
that may surround
DNA to increase its
processivity.
11,DNA polymerase III and many other
proteins are part of the replisome for
DNA replication in E.coli cells
? In vitro studies revealed about 20 proteins are
involved in DNA replication in E.coli cells.
? These include,
– the helicase (dnaB) for moving along the DNA
and separating the two DNA strands using energy
from ATP;
– the topoisomerases for relieving topological
(torsional) strains in the helical structure (positive
supercoils) generated during strand separation;
– the DNA-binding protein for binding and
stabilizing the single stranded DNA generated;
– the primase (dnaG) for generating a short RNA
primer;
– the DNA polymerase III for polymerizing and
proofreading the nucleotides according to the
templates;
– the DNA polymerase I (polA) for removing the
RNA primers and replacing by a DNA sequence;
– the DNA ligase for sealing nicks between the
Okazaki fragments.
12,A 245 bp fragment in the E.coli
chromosomal DNA,OriC,was identified
to be the replication origin
? It was identified by using origin-lacking plasmids.
? OriC contains a tandem array of three 13-mer with nearly
identical sequences and four 9-mer repeats,all of which
highly conserved among all bacterial replication origins.
? OriC contains 9 GATC (palindromic) sequences,which can
be methylated at the base ring of A,which is believed to be
important in having only one replication per cell division.
OriC,the replication origin of E,coli
Chromosome,contains three repeats
Of 13 bp ad four repeats of 9 bp.
GATC 5GATC
13,The molecular details of the
replication of E,coli chromosomal DNA
are the best understood by in vitro studies
? The whole process can be divided into three stages,
initiation,elongation,and termination.
? During the initiation stage,the DNA is open at the
OriC site and a prepriming complex is formed,with
the participation of at least nine proteins.
? During the elongation stage,short RNA primers are
synthesized by the primase,extended by the DNA
polymerase III,and ligated by the DNA ligase (for
the Okazaki fragments of the lagging strand).
? During the termination stage,the two replication
forks meet at the Ter sequences,the replisome
dissociates,and the two catenated chromosomes are
separated by DNA topoisomerase IV.
14,Multiple proteins participate at the
initiation stage of DNA replication of the
E,coli chromosome
? The replication process begins when a single
complex of about 20 DnaA protein molecules (with
ATP bound) binds to the four 9 bp repeats at OriC
that is supercoiled,
? Aided by HU (a histone-like protein),the bound
DnaA complex opens the two DNA strands at the
AT-rich 13-mer repeats.
? DnaB,the helicase,binds (aided by DnaC protein)
to the opened DNA strands as two hexamer clamps
and further opens (unwinds) the strands in both
directions,thus forming the prepriming complex.
The prepriming complex is
formed at the OriC,with the
participation of DnaA,HU,
DnaB,DnaC and other
proteins.
The prepriming
complex
15,The leading strand is synthesized
continuously but the lagging strand
discontinuosuly
? Further unwinding of the DNA duplex needs DNA
gyrase ( a topoisomerase II) to relieve the supercoils
ahead the replication forks and SSB for stabilizing
the unwound single strand DNAs.
? Kornberg discovered that the nascent DNA is
always covalently linked to a short stretch of RNA.
? The RNA primers were found to be 10-60
nucleotides in length.
? The primase (a RNA polymerase),recruited to the
open templates via DnaB,was found to be the
enzyme that catalyzes the synthesis of the RNA
primers.
? For the leading strand,only one primer is
synthesized,which is then elongated by DNA
polymerase III in a continuous way.
? On the lagging strand,DnaB (the helicase)
intermittently recruits DnaG (the DNA primase) to
form a complex called primosome,and repeatedly
making primers for the Okazaki fragments,
? Each Okazaki fragment is then synthesized on one
RNA primer by DNA polymerase III.
? The RNA primers are removed and replaced by a
DNA sequence in a reaction catalyzed by DNA
polymerase I,using its nick translation activity (5`
to 3` exonuclease + polymerase).
? The final Okazaki fragments are joined together by
the DNA ligase.
? DNA ligase catalyzes the formation of a
phosphodiester bond between a 3`-OH group and a
5` phosphate group of two DNA strands,where the
5` phosphate is first activated by being modified by
an AMP group coming from NAD+ (the bacterial
enzyme) or ATP (the virus and animal enzymes),
Only one RNA primer is needed for
synthesizing the leading strand.
(DnaB)
RNA primers are
repeatedly formed
by the primase on
the lagging strand.
DNA polymerase I replaces the RNA primers by
DNA sequences and DNA ligase seals the nicks
DNA ligase first transfers
an AMP moiety to the 5`
phosphate group (from
NAD+ or ATP) before
making a phosphodiester
bond.
16,The leading and lagging strands are
believed to be synthesized coordinately by
a single asymmetric DNA polymerase III
dimer
? The two polymerase III cores (having the ae?
subunits) are believed to be connected by two t
subunits.
? The coupling is believed to accomplish via looping
of the lagging strand template.
? The PolIII on the lagging strand intermittently
releases the b sliding clamp and the completed
Okazaki fragment before rebinding to a new b
subunit dimer loaded at a RNA primer and
synthesizing a new Okazaki fragment.
? Accumulating evidence seem to support that the Pol
III dimer complex is associated with the plasma
membrane and does not actually itself move; the
DNA moves through the fixed complex.
Sliding clamp
subunits
Catalytic
subunit
Catalytic
subunit
3’ 5’ exonuclease
subunits
Clamp
loader
Proposed
architecture of DNA
polymerase III
holoenzyme,
an asymmetric
dimer
No 5` to 3` exonuclease
activity
17,E.coli DNA replication ends at a
specific terminus region with multiple
copies of a 20 bp Ter sequence
? The Ter sequences are positioned in two clusters
with opposite orientations.
? The Tus protein can bind to the Ter sequences.
? At each round of DNA replication,only one Tus-Ter
complex seem to function to arrest a replication fork
from one direction,with the opposing replication
fork stopping when the two collide.
? The Ter sequences do not seem to be essential for
stopping replication.
? The two newly synthesized circular chromosomal
DNAs are topologically interlinked (catenated) and
is finally separated by the action of a Type II
topoisomerases,
18,Methylation of GATC sequences at
oriC is believed to control the replication
frequencies in E,coli cells
? Hemimethylated oriC was found unable to initiate
another round of DNA replication.
? It is believed that the hemimethylated GATC sequences
at OriC is sequestered in the plasma membrane
immediately after one round of DNA replication,thus
blocking another round of replication until the GATC
sequences are released and fully methylated by Dam
methylase.
? The delay of methylation is hypothesized to limit DNA
replication to occur once per cell division,
? The slow hydrolysis of ATP by DnaA protein was also
proposed to regulate replication initiation.
Hemimethylatd OriC
is not active for
initiating DNA
replication
Active
Inactive
19,DNA replication in eukaryotic cells
use essentially the same principles but
more complex in the details
? The best understood eukaryotic replication system is
that of SV40 and yeast.
? Formation of the RNA primers and the initial
incorporation of deoxynucleotides are believed to be
catalyzed by DNA polymerase a (which has no
proofreading activity).
? Later chain extension is believed to be catalyzed by
DNA polymerase d,which has proofreading activity
and is
? Specific sequences (~150 bp) that can function as
replication origins have only been identified in yeast
( is called autonomously replicating sequences,or
ARS) and SV40 virus.
? The rate of DNA synthesis in eukaryotic cells is
about one tenth of that of the E.coli cells.
? Replication in the eukaryotic genomes begin at
many replication origins.
? 9 purified proteins are needed to replicate SV40
virus DNA in vitro.
? T antigen--a multifunctional site-specific DNA
binding protein encoded by SV40 DNA,binds to the
origin (as DnaA) and melts the duplex DNA (as
DnaB);
? RPA– encoded by the host mammalian cells and binds
to the melted single-stranded DNA (as SSB).
? Pol a/primase-- synthesizes the RNA primers and a
stretch of DNA sequences,has no proofreading activity.
? Pol d– replaces Pol a/primase to further extend the
RNA-DNA strand,has proofreading activity,
? PCNA (proliferating cell nuclear antigen)-- a trimeric
ring-shaped protein that clamps Pol d onto the DNA
template.
? RFC (replication factor C)-- a clamp loader for PCNA,
Topoisomerases-- Relieves the torsional strain induced
by the growing replication fork.
? Ligases--joins the Okazaki fragments (which is much
shorter than those in E.coli cells),as well as the leading
strand.
Model of in vitro replication
of SV40 DNA by eukaryotic
enzymes:
1,T antigen (Tag) binds and
unwinds replication origin;
2,RPA (or RFA) binds to
single-stranded DNA;
3,Pol a-primase
synthesizes the primers
(RNA + DNA).
4,RFC loads PCNA to the
template.
5,PCNA displaces
Pola-primase and
functions as a
DNA clamp;
6,Pol d replaces
Pola-primase
and further
extends the DNA
strands.
20,Maintaining the integrity of the
genomic DNA is essential to all cells
? The DNA molecules can become damaged via a
variety of internal (e.g.,autonomous deamination of
bases) or external (e.g.,exposure to UV light and
chemical agents) processes.
? A diversity of repair systems have evolved,
mismatch repair,base-excision repair,nucleotide-
excision repair and direct repair are the common
types found.
? Many DNA repair systems are energetically
expensive (very inefficient comparing with other
metabolic pathways) and redundant (especially to
some common types of lesions).
? Unrepaired DNA damages can introduce mutations,
which can cause cancers in mammals (e.g.,
xeroderma pigmentosum,着色性干皮病,is caused
by a defect of the DNA repair systems).
? Most carcinogens are found to be strong mutagens,
the Ames Test has been used to examine whether a
chemical is a potential human carcinogen by testing
its mutagenicity on certain bacterial strains.
? Complementary synthesis (based on the duplex
DNA structure) after elimination of the damaged
nucleotides is a common principle for all the repair
systems,
Ames Test:
carcinogens are
tested for their
mutagenicity on
bacterial genomes.
Plating of
his- salmonella
typhimurium
High level of mutagen
Medium level of mutagen Low level of mutagen
No mutagen Back
mutations
21,The mismatch repair pathway acts to
increase the fidelity of DNA replication
? In vitro studies showed that the transient
undermethylation of GATC sequences in the newly
synthesized strand permits strand discrimination in
E.coli (not in eukaryotic cells),mismatches are
corrected according to information provided by the
parent strand.
? At least 12 proteins are involved in mismatch repair in
E,coli,among which the MutS-MutL complex acts to
recognize all the mismatches (except C-C) and MutH
acts to recognized GATC sequences and generates a
nick on the 5` side of the G in the (5`) GATC in the
newly synthesized (unmethylated) strand when the two
meet.
? Exonucleases (I or X cleaves from 3` to 5`direction;
VII or RecJ cleaves from 5` to 3` direction) remove
a segment of DNA from the newly synthesized
strand including the mispaired nucleotide.
? DNA pol III resynthesizes the removed segment.
? DNA ligase reseals the gap on the newly
synthesized DNA.
The Mut L,MutS,and MutH
proteins in E,coli recognize
the mismatched base pairs
and generate a nick at a
GATC sequence up to 1kb
away.
MutL links MutS (recognizing the
mismatch and MutH (recognizing
the GATC sequence)
MutS
A proposed model for methyl-directed
mismatch repair in E,coli cells.
Mismatch on the 3` side
of the cleavage site Mismatch on the 5` side of the cleavage site
22,Base-excision repair acts by
removing the damaged bases first
? The key enzymes for this class of repair include the
specific DNA glycosylases (e.g.,uracil,hypoxanthine,
3-methyladenine,7-methylguanine,and pyrimidine
dimers glycosylases) and the nonspecific AP
(apurinic or apyrimidinic) endonucleases.
? The DNA glycosylases remove specific DNA lesions
by cleaving the N-glycosyl bonds.
? AP endonucleases cleave the phosphodiester bond
near the AP site (either on the 5` or the 3` side of AP).
? DNA polymerase I replaces a segment of DNA
including the AP site and DNA ligase seals the gap.
The proposed model
for base-excision
repair using specific
glycosylases and
nonspecific AP
endonucleases.
23,Nucleotide-excision repair acts by
removing a short fragment of ssDNA
containing the lesion
? Usually works when the DNA lesion (e.g.,the
presence of cyclobutane pyrimidine dimers or 6-4
photoproducts) causes large distortion in the helical
structure and is probably the most important way for
DNA repair in cells.
? in E,coli,ABC exinuclease,a complex of three
proteins (UvrA,UvrB,and UvrC),scans the lesion
(A2B),generates two cuts (3` side by B and 5` side by
C) on the two sides of the damaged site.
? A 12 mer segment of DNA spanning the damaged
bases is released by the UvrD helicase.
? DNA Pol I fills the gap and DNA ligase seals it.
? The exinuclease in eukaryotic cells contains 16
polypeptides,with none homologous to that of the
E.coli enzyme complex.
? A 29 mer is released by the human exinuclease.
24,Direct repair acts by directly reverse
the base modifications without removing
a base or nucleotide
? Pyrimidine dimers can be directly converted to two free
monomeric pyrimines in a reaction catalyzed by DNA
Photolyases via free radical intermediates.
? The methyl group of O6-methylguanine (introduced by
alkylating agents) is directly transferred to a Cys
residue on O6-methylguanine-DNA
methyltransferase (not an enzyme in strict sense).
? The methylated O6-methylguanine-DNA
methyltransferase act as a transcription activator for its
own gene and a few other genes also encoding repair
enzymes.
A proposed action
mechanism for the
photolyases
Chromophores
Three unstable radicals
O6-methylguanine-DNA
methyltransferase
Cys-S CH3
O6 methylguanine on DNA is
directely repaired by removing
the methyl group by a Cys
residue of the methyltranseferase
A transcription
activator
25 Recombinational repair or error-
prone repair have to occur when the
complementary strand is also damaged
or absent
? These types of lesions include double-strand breaks,
double strand cross-links,both complementary
strand are damaged or one is damaged with the other
absent (as will occur when a replication fork
encounters an unrepaired lesiono r strand break).
? The lesions can be repaired by using information
from a separate,homologous chromosome via
recombinational DNA repair (discussed later).
? When the DNA strands are extensively damaged,the
SOS response and error-prone repair (or translesion
replication,being a desperate strategy) will occur.
? Some proteins may act to repair lesions (the ABC
exinuclease,RecA,Pol II),some to inhibit cell divisions
(e.g.,sulA),and others to act in translesion replication
(Rec A protein,SSB,DNA polymerase V made of
UmuC and the shortened UmuD or DNA polymerase IV
encoded by the dinB gene).
? Random nucleotides may be added in place of the
damaged ones during translesion replications,but the
detailed mechanism has not been revealed.
? The increased mutations may generate a few cells
(although almost all dead) that are better fit to survive
the catastrophic conditions (better species may thus
evolve).
When a replication fork passes the unrepaired lesions
situations will be generated where both complementary
strands of a duplex are unable to act as a template.
26,DNA rearranges in cells via
genetic recombination
? Blocks of genes from homologous chromosomes
(e.g.,between homologous paternal and maternal
chromosomes) were found to be exchanged by the
process of crossing over,or homologous
recombination during Drosophila meiosis (early
20th century).
? Homologous recombination was then found to occur
commonly in all types of organisms between two
DNA sequences sharing homology.
? Two more major types of DNA rearrangements were
later revealed.
? Site-specific recombination was found to occur
when the DNA of l phage integrates into the E.coli
genome,where the DNA exchange occurs only
between specific DNA sequences.
? Site-specific recombination was also found to occur
in rearranging certain genes in all types of
eukaryotic cells.
? Certain DNA elements,called transposons,were
found to be able to randomly transpose (mobilize,
hop) from one location to another on chromosomes,
thus called DNA transposition (speculated by
Barbara McClintock while studying maize genetics
in the 1950s),which was also found in bacteria and
animals many years later.
? All the strands of the recombining DNA molecules
have to be broken and rejoined with new strands for
all these recombinations.
? Novel DNA intermediates with unusual structures
(with three or four strands interwound) have to be
formed.
? Principles of the three kinds of recombinations have
been widely used to carry out artificial
manipulations of DNAs (or genetic engineering) in
all types of cells.
Homologous recombination is believed to occur
between the closely associated chromatids (in tetrads)
during the prophase of the first meiotic division of
germ-line cells to produce haploid gametes.
Current studies show that these observed
“crossover” sites may not promote
but inhibit DNA exchange.
EM examination of a chiasmata,no contact
between homologous DNA molecules!
