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 (only two
types would be expected if replication is
conservative),
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 autoradiographs 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 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 (using radioactive
precursor nucleotides to label and trichloroacetic
acid to precipitate the newly synthesized DNA).
? 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 is a 103-kDa
trifunctional protein having one
polymerase and two 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 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 is unique for DNA
polymerase I,enabling 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
(clean-up functions),also for incorporating
radioisotope-labeled dNTPs into a DNA probe (in
vitro labeling).
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
68 kDa35 kDa
The 3` to 5`
exonuclease
domain
The polymerase
domain
Structure of the
Klenow fragment
of DNA polymease I
The 5` to 3` exonuclease
and polymerase activities
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 in vivo.
? 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
discovered in E.coli cells in the early 1970s (~15 years
after Pol I was discovered!) 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
complex containing at least 10 different subunits.
? The holoenzyme seems to exist 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 dimer provides high processivity,with more
than 500,000 nucleotides added per binding.
? The rate of DNA synthesis is high,with 1000
nucleotides/second (only about 20 for Pol I).
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 (unwinding) 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 Single-stranded DNA-binding protein (SSB)
for binding and stabilizing separated DNA strands;
– the primase (dnaG) for generating a short RNA
primer;
– The multimeric 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 it by a DNA sequence;
– the DNA ligase for sealing nicks.
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 regulating the
frequency of DNA replication,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
first synthesized by the primase,which is then
extended by the DNA polymerase III,and finally
ligated by the DNA ligase.
? 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 (negatively) 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 (positive)
supercoils generated ahead the replication forks and
SSB for stabilizing the unwound single strand DNAs.
? RNA synthesis was found to be needed for DNA
replication.
? 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 (the helicase),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 intermittently recruits
DnaG (the primase) to form a complex called
primosome (引发体 ),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 at a nicked DNA duplex,where
the 5` phosphate is first activated by being modified
by an AMP group (adenylylation) coming from
NAD+ (for the bacterial enzyme ligase) or ATP (for
the virus and animal ligases),
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 Pol III 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 then
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,
Replication terminators may help halt the replication
forks,but seems to be not essential for stopping DNA
replication in E,coli.
The newly synthesized
two chromosomal
DNA molecules are
interlinked (catenated)
And are separated by
DNA topoisomerase IV.
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
(DNA adenine methylation) 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
seems to be sequenstered
in the plasma membrane,
and thus being not active for
initiating DNA replication
Active Inactive
19,DNA replication in eukaryotic cells
use essentially the same principles but
being 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 clamped onto the DNA templates via the
PCNA trimer rings.
? 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!
27,Homologous DNA recombinations
during meiosis is believed to be initiated
with double-strand breaks
? The two homologous DNA molecules,with one
having a double-stranded break,have to be first
aligned.
? Two 3` single strand extensions are then generated
by the action of an exonuclease.
? Two crossover structures called Holliday
intermediates are then formed by the invasion of
the exposed 3` ends in the intact duplex DNA
followed by branch migration and replication,
resulting in a region of heteroduplex DNA.
? Four (two for each duplex) cleavages of the
Holliday intermediates followed by rejoining result
in either two nonrecombinant or recombinant DNA
duplexes.
? Holliday intermediate was directly observed
between plasmid and virus DNA molecules.
? Such homologous recombination leads to the
generation of genetic diversity,orderly segregation
of chromosomes,or repair of several types of DNA
damages.
Isomeric Holliday
intermediates between
two plasmids
Single-stranded
ring
28,Proteins involved in homologous
recombination have been identified in
E.coli
? RecBCD enzyme (encoded by three genes),
– Able to bind to a free blunt end of a DNA (double
strand breaks) and travel along the duplex with its
helicase activity unwinding the duplex,
meanwhile degrading both single strands using its
dual 5’ 3’ and 3’ 5’ exonuclease activities.
– when RecBCD encounters a chi sequence,it
generates a 3` single-stranded extensions (A total
of 1009 chi sequences have been revealed in E,
coli).
? RecA protein,able to form nucleoprotein filament
on single-stranded DNA or duplex DNA with a
single-stranded gap in vitro; able to promote strand
exchange,probably via a spooling action,between
three or four homologous DNA strands in vitro; is
believed to mediate strand exchange and branch
migration to form the Holliday intermediate in vivo.
? RuvC,the resolvase,cleaves the Holliday
intermediate to generate recombinant or
nonrecombinant products.
? Topoisomerases,DNA polymerases and DNA
ligases also act during DNA recombination.
? Much more studies are needed to fully understand
homologous recombination.
The RecBCD enzyme binds to
a double stran DNA break,
cleaves both strands,generates
a 3` single strand extension
When meeting a chi sequence
with a free –OH group.
5` GCTGGTGG 3`
EM picture and
computer imaging
model of a
nucleoprotein filament
formed between RecA
and a single-
stranded DNA.
29,DNA lesions unrepaired will stall the
replication process and are believed to be
repaired via homologous recombination
before replication restarts
? A lesion in a single-strand gap is repaired via a
process requiring RecA,RecF,RecO,and RecR.
? A double strand break is repaired via a process
requiring RecA and RecBCD.
? After fixation,replication restarts using the
replication restart primosome.
? All three aspects of DNA metabolism work together
to repair halted replication forks!
Replication forks stall at
unrepaired lesions,which is
then fixed via homologous
recombination before
replication restarts via the
replication restart primosome.
30,Site-specific recombinations occur
only between specific DNA sequences of
20-200bp and are catalyzed by specific
recombinases
? Site-specific recombinations have specialized
functions (including virus integration into host
genomes; regulation of expression of certain genes;
programmed DNA rearrangements during cell
differentiation).
? In vitro studies of different site-specific
recombination systems seem to suggest a common
reaction pathway,
? First a specific (tetrameric) recombinase binds to two
specific DNA sites with the short nonpalindromic
sequences aligned in the same orientation.
? Short stretches of identical sequences are usually shared
by the two sites.
? One DNA strand in each DNA duplex is cleaved at a
specific point,forming two transient covalent
intermediates between the DNA and the recombinase
(similar to what happens when topoisomerases act).
? The two free ends at the cleaved site are then exchanged
and joined to a new partner,generating a Holliday
intermediate.
? An isomerization of the Holliday intermediate switches
the position of the newly joined strands and the yet-cut
strands (accomplished by a rotation of the recombinase).
? Another round of chain breakage and rejoining
completes the whole recombination process.
? In some systems,both strands of each
recombining DNA segment are cut concurrently
and rejoined to new partners (Holliday
intermediate would not be formed in these cases).
? The chains exchange reciprocally and precisely
during site-specific recombination.
? The recombinase acts as site-specific
endonuclease,as well as DNA ligase.
31,A site-specific recombination leads to
DNA inversion,deletion or insertion
depending on the location and
orientation of the recombining sites
? When the two recombination sites are located on the
same DNA molecule,an opposite orientation leads
to inversion of the intervening DNA fragment; a
common orientation leads to deletion.
? When the two recombination sites are located on
two different DNA molecules,insertion will be
generated if one or two of the DNA molecules are
circular.
Site-specific recombination on the same DNA
molecule leads to either inversion or deletion of
the intervening DNA fragments.
32,Bacteriophage l DNA can be
integrated into and excised from E.coli
genomic DNA via site-specific
recombination
? This is the first site-specific recombination system
identified and studied in vitro.
? The integration is accomplished by a phage-
encoded recombinase called the l integrase,
where exchange of single strands probably take
place sequentially.
? Excision of the phage DNA from the bacterial
genomic DNA needs auxiliary proteins other than
the recombinase.
Integration of bacteriophage l DNA into E.coli genomic DNA via
site-specific recombination
1,The recombinase (l integrase) is encoded by the phage DNA;
2,The recombination sites (attP on the phage DNA,attB on the
bacterial DNA) share 15 bp of complete homology;
3,Excision of the l DNA from the host genomic DNA occurs when
the host cells are under stress conditions,using different
recombining sites and different auxiliary proteins (FIS and XIS,
encoded by the bacterium and phage respectively.
15 bp
33,The large number of different
antibodies are generated via site-specific
recombinations in vertebrates
? A human can generate about 108 different antibody
proteins,each having different binding specificities.
? The whole human genome consists of about 4 X104
genes.
? This antibody diversity was found (in 1976 by
Tonegawa) to be generated from site-specific
recombination during the development of antibody-
generating mature B cells from stem cells.
? Segments of DNA are randomly joined together via
site-specific recombination to form the millions of
genes encoding the light and heavy chains of the 108
different antibodies.
? RAG (recombination activating gene) proteins
catalyzes the formation of double strand breaks and
hairpin ends between the recombination signal
sequences (RSS) and the V or J segment.
? The V and J segements are then joined (with
variations) by the action of a second complex of
proteins.
Programmed DNA
deletions resulted
from site-specific
recombinations
lead to the diversity
of immunoglobulins.
A proposed mechanism
on the removal of the
intervening DNA between
a V and a J segment
34,Transposable genetic elements move
from one location to another via
? DNA transposition was originally speculated by
Barbara McClintock (1940s) while studying maize
genetics,later found to be present in all types of
cells;
? DNA transposition occurs randomly,but very
infrequently.
? The transposable genetic elements can be divided
into two families,transposons move directly as a
DNA fragment and retrotransposons move via a
RNA intermediate,sharing similarity to retroviruses,
The two major
classes of mobile
DNA elements
35,DNA transposition in bacteria are
either direct or replicative
? There are two classes of transposons in bacteria
? Insertion sequences,being the simple transposons
containing only the sequences required for their
transposition and the genes for transposases that
promote the processes.
? Complex transposons,contain additional genes that
often code for proteins confer resistance to antibiotics
(being one source of generating drug-resistance
bacteria).
? Most bacterial transposons have short repeats at the
two ends,serving as binding sites for the transposases.
? A short sequence at the target site (5-10 bp) is
always duplicated (flanking each end of the
inserted transposons),reflecting the cutting
mechanism used to insert a transposon into the
target site.
? DNA transposition in bacteria can be either
direct (leaving a double strand at the donor
DNA) or replicative (leaving the donor
transposon intact)
The staggered cuts
generated by the
transposases at the
target sites lead to
the duplication of short
target sequences at the
two ends of the inserted
transposons,
DNA transposition in
bacteria can be either
direct or replicative.
Summary
? DNA replication begins at specific origins,is
semiconservative,bidirectional,and
semidiscontinuous.
? All DNA polymerases need a primer,extend the
DNA chain in 5` to 3` direction (using dNTPs),and
have 3` to 5` exonuclease activity for proofreading.
? DNA polymerase III in E,coli,DNA polymerase a
and d in eukayrotic cells are responsible for DNA
relication in vivo.
? DNA replisomes contain many protein components
including helicases,single-stranded DNA binding
proteins,topoisomerases,primases,ligases etc.
? It is likely that one DNA polymerase complex
catalyzes the synthesis of both the leading and
lagging strands at each replication fork via DNA
looping.
? Complex and redundant DNA repair systems have
evolved to correct lesions in the DNA molecules.
? The multiple DNA repair systems include mismatch
repair (using Mut L,MutS,and MutH proteins in
E.coli),base-excision repair (using specific DNA
glycosylases and nonspecific AP endonucleases in E,
coli),nucleotide-excision repair (using a
excinuclease complex),direct repair (using specific
enzymes or other proteins),homologous repair,and
error-prone repair (use under desperate conditions).
? DNA recombinations include different types,
homologous,site-specific,and transposition.
? Homologous recombination occurs between any two
homologous sequences,and is believed to be
initiated by double-strand breaks,with RecBCD
(helping to generate a 3`-terminal single-stranded
end near the chi sequences),RecA (act to promote
strand exchange and branch migration),RuvC (to
resolve the Holliday structure) actively involved in
E,coli.
? Site-specific recombinations occur between DNA
molecules (or regions) having specific sequences
and is catalyzed by specific recombinases.
? Transposable genetic elements,found in all types of
organisms,jumps from one chromosomal location to
another via either direct transposition or replicative
transposition.
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 (only two
types would be expected if replication is
conservative),
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 autoradiographs 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 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 (using radioactive
precursor nucleotides to label and trichloroacetic
acid to precipitate the newly synthesized DNA).
? 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 is a 103-kDa
trifunctional protein having one
polymerase and two 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 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 is unique for DNA
polymerase I,enabling 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
(clean-up functions),also for incorporating
radioisotope-labeled dNTPs into a DNA probe (in
vitro labeling).
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
68 kDa35 kDa
The 3` to 5`
exonuclease
domain
The polymerase
domain
Structure of the
Klenow fragment
of DNA polymease I
The 5` to 3` exonuclease
and polymerase activities
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 in vivo.
? 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
discovered in E.coli cells in the early 1970s (~15 years
after Pol I was discovered!) 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
complex containing at least 10 different subunits.
? The holoenzyme seems to exist 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 dimer provides high processivity,with more
than 500,000 nucleotides added per binding.
? The rate of DNA synthesis is high,with 1000
nucleotides/second (only about 20 for Pol I).
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 (unwinding) 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 Single-stranded DNA-binding protein (SSB)
for binding and stabilizing separated DNA strands;
– the primase (dnaG) for generating a short RNA
primer;
– The multimeric 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 it by a DNA sequence;
– the DNA ligase for sealing nicks.
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 regulating the
frequency of DNA replication,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
first synthesized by the primase,which is then
extended by the DNA polymerase III,and finally
ligated by the DNA ligase.
? 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 (negatively) 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 (positive)
supercoils generated ahead the replication forks and
SSB for stabilizing the unwound single strand DNAs.
? RNA synthesis was found to be needed for DNA
replication.
? 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 (the helicase),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 intermittently recruits
DnaG (the primase) to form a complex called
primosome (引发体 ),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 at a nicked DNA duplex,where
the 5` phosphate is first activated by being modified
by an AMP group (adenylylation) coming from
NAD+ (for the bacterial enzyme ligase) or ATP (for
the virus and animal ligases),
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 Pol III 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 then
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,
Replication terminators may help halt the replication
forks,but seems to be not essential for stopping DNA
replication in E,coli.
The newly synthesized
two chromosomal
DNA molecules are
interlinked (catenated)
And are separated by
DNA topoisomerase IV.
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
(DNA adenine methylation) 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
seems to be sequenstered
in the plasma membrane,
and thus being not active for
initiating DNA replication
Active Inactive
19,DNA replication in eukaryotic cells
use essentially the same principles but
being 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 clamped onto the DNA templates via the
PCNA trimer rings.
? 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!
27,Homologous DNA recombinations
during meiosis is believed to be initiated
with double-strand breaks
? The two homologous DNA molecules,with one
having a double-stranded break,have to be first
aligned.
? Two 3` single strand extensions are then generated
by the action of an exonuclease.
? Two crossover structures called Holliday
intermediates are then formed by the invasion of
the exposed 3` ends in the intact duplex DNA
followed by branch migration and replication,
resulting in a region of heteroduplex DNA.
? Four (two for each duplex) cleavages of the
Holliday intermediates followed by rejoining result
in either two nonrecombinant or recombinant DNA
duplexes.
? Holliday intermediate was directly observed
between plasmid and virus DNA molecules.
? Such homologous recombination leads to the
generation of genetic diversity,orderly segregation
of chromosomes,or repair of several types of DNA
damages.
Isomeric Holliday
intermediates between
two plasmids
Single-stranded
ring
28,Proteins involved in homologous
recombination have been identified in
E.coli
? RecBCD enzyme (encoded by three genes),
– Able to bind to a free blunt end of a DNA (double
strand breaks) and travel along the duplex with its
helicase activity unwinding the duplex,
meanwhile degrading both single strands using its
dual 5’ 3’ and 3’ 5’ exonuclease activities.
– when RecBCD encounters a chi sequence,it
generates a 3` single-stranded extensions (A total
of 1009 chi sequences have been revealed in E,
coli).
? RecA protein,able to form nucleoprotein filament
on single-stranded DNA or duplex DNA with a
single-stranded gap in vitro; able to promote strand
exchange,probably via a spooling action,between
three or four homologous DNA strands in vitro; is
believed to mediate strand exchange and branch
migration to form the Holliday intermediate in vivo.
? RuvC,the resolvase,cleaves the Holliday
intermediate to generate recombinant or
nonrecombinant products.
? Topoisomerases,DNA polymerases and DNA
ligases also act during DNA recombination.
? Much more studies are needed to fully understand
homologous recombination.
The RecBCD enzyme binds to
a double stran DNA break,
cleaves both strands,generates
a 3` single strand extension
When meeting a chi sequence
with a free –OH group.
5` GCTGGTGG 3`
EM picture and
computer imaging
model of a
nucleoprotein filament
formed between RecA
and a single-
stranded DNA.
29,DNA lesions unrepaired will stall the
replication process and are believed to be
repaired via homologous recombination
before replication restarts
? A lesion in a single-strand gap is repaired via a
process requiring RecA,RecF,RecO,and RecR.
? A double strand break is repaired via a process
requiring RecA and RecBCD.
? After fixation,replication restarts using the
replication restart primosome.
? All three aspects of DNA metabolism work together
to repair halted replication forks!
Replication forks stall at
unrepaired lesions,which is
then fixed via homologous
recombination before
replication restarts via the
replication restart primosome.
30,Site-specific recombinations occur
only between specific DNA sequences of
20-200bp and are catalyzed by specific
recombinases
? Site-specific recombinations have specialized
functions (including virus integration into host
genomes; regulation of expression of certain genes;
programmed DNA rearrangements during cell
differentiation).
? In vitro studies of different site-specific
recombination systems seem to suggest a common
reaction pathway,
? First a specific (tetrameric) recombinase binds to two
specific DNA sites with the short nonpalindromic
sequences aligned in the same orientation.
? Short stretches of identical sequences are usually shared
by the two sites.
? One DNA strand in each DNA duplex is cleaved at a
specific point,forming two transient covalent
intermediates between the DNA and the recombinase
(similar to what happens when topoisomerases act).
? The two free ends at the cleaved site are then exchanged
and joined to a new partner,generating a Holliday
intermediate.
? An isomerization of the Holliday intermediate switches
the position of the newly joined strands and the yet-cut
strands (accomplished by a rotation of the recombinase).
? Another round of chain breakage and rejoining
completes the whole recombination process.
? In some systems,both strands of each
recombining DNA segment are cut concurrently
and rejoined to new partners (Holliday
intermediate would not be formed in these cases).
? The chains exchange reciprocally and precisely
during site-specific recombination.
? The recombinase acts as site-specific
endonuclease,as well as DNA ligase.
31,A site-specific recombination leads to
DNA inversion,deletion or insertion
depending on the location and
orientation of the recombining sites
? When the two recombination sites are located on the
same DNA molecule,an opposite orientation leads
to inversion of the intervening DNA fragment; a
common orientation leads to deletion.
? When the two recombination sites are located on
two different DNA molecules,insertion will be
generated if one or two of the DNA molecules are
circular.
Site-specific recombination on the same DNA
molecule leads to either inversion or deletion of
the intervening DNA fragments.
32,Bacteriophage l DNA can be
integrated into and excised from E.coli
genomic DNA via site-specific
recombination
? This is the first site-specific recombination system
identified and studied in vitro.
? The integration is accomplished by a phage-
encoded recombinase called the l integrase,
where exchange of single strands probably take
place sequentially.
? Excision of the phage DNA from the bacterial
genomic DNA needs auxiliary proteins other than
the recombinase.
Integration of bacteriophage l DNA into E.coli genomic DNA via
site-specific recombination
1,The recombinase (l integrase) is encoded by the phage DNA;
2,The recombination sites (attP on the phage DNA,attB on the
bacterial DNA) share 15 bp of complete homology;
3,Excision of the l DNA from the host genomic DNA occurs when
the host cells are under stress conditions,using different
recombining sites and different auxiliary proteins (FIS and XIS,
encoded by the bacterium and phage respectively.
15 bp
33,The large number of different
antibodies are generated via site-specific
recombinations in vertebrates
? A human can generate about 108 different antibody
proteins,each having different binding specificities.
? The whole human genome consists of about 4 X104
genes.
? This antibody diversity was found (in 1976 by
Tonegawa) to be generated from site-specific
recombination during the development of antibody-
generating mature B cells from stem cells.
? Segments of DNA are randomly joined together via
site-specific recombination to form the millions of
genes encoding the light and heavy chains of the 108
different antibodies.
? RAG (recombination activating gene) proteins
catalyzes the formation of double strand breaks and
hairpin ends between the recombination signal
sequences (RSS) and the V or J segment.
? The V and J segements are then joined (with
variations) by the action of a second complex of
proteins.
Programmed DNA
deletions resulted
from site-specific
recombinations
lead to the diversity
of immunoglobulins.
A proposed mechanism
on the removal of the
intervening DNA between
a V and a J segment
34,Transposable genetic elements move
from one location to another via
? DNA transposition was originally speculated by
Barbara McClintock (1940s) while studying maize
genetics,later found to be present in all types of
cells;
? DNA transposition occurs randomly,but very
infrequently.
? The transposable genetic elements can be divided
into two families,transposons move directly as a
DNA fragment and retrotransposons move via a
RNA intermediate,sharing similarity to retroviruses,
The two major
classes of mobile
DNA elements
35,DNA transposition in bacteria are
either direct or replicative
? There are two classes of transposons in bacteria
? Insertion sequences,being the simple transposons
containing only the sequences required for their
transposition and the genes for transposases that
promote the processes.
? Complex transposons,contain additional genes that
often code for proteins confer resistance to antibiotics
(being one source of generating drug-resistance
bacteria).
? Most bacterial transposons have short repeats at the
two ends,serving as binding sites for the transposases.
? A short sequence at the target site (5-10 bp) is
always duplicated (flanking each end of the
inserted transposons),reflecting the cutting
mechanism used to insert a transposon into the
target site.
? DNA transposition in bacteria can be either
direct (leaving a double strand at the donor
DNA) or replicative (leaving the donor
transposon intact)
The staggered cuts
generated by the
transposases at the
target sites lead to
the duplication of short
target sequences at the
two ends of the inserted
transposons,
DNA transposition in
bacteria can be either
direct or replicative.
Summary
? DNA replication begins at specific origins,is
semiconservative,bidirectional,and
semidiscontinuous.
? All DNA polymerases need a primer,extend the
DNA chain in 5` to 3` direction (using dNTPs),and
have 3` to 5` exonuclease activity for proofreading.
? DNA polymerase III in E,coli,DNA polymerase a
and d in eukayrotic cells are responsible for DNA
relication in vivo.
? DNA replisomes contain many protein components
including helicases,single-stranded DNA binding
proteins,topoisomerases,primases,ligases etc.
? It is likely that one DNA polymerase complex
catalyzes the synthesis of both the leading and
lagging strands at each replication fork via DNA
looping.
? Complex and redundant DNA repair systems have
evolved to correct lesions in the DNA molecules.
? The multiple DNA repair systems include mismatch
repair (using Mut L,MutS,and MutH proteins in
E.coli),base-excision repair (using specific DNA
glycosylases and nonspecific AP endonucleases in E,
coli),nucleotide-excision repair (using a
excinuclease complex),direct repair (using specific
enzymes or other proteins),homologous repair,and
error-prone repair (use under desperate conditions).
? DNA recombinations include different types,
homologous,site-specific,and transposition.
? Homologous recombination occurs between any two
homologous sequences,and is believed to be
initiated by double-strand breaks,with RecBCD
(helping to generate a 3`-terminal single-stranded
end near the chi sequences),RecA (act to promote
strand exchange and branch migration),RuvC (to
resolve the Holliday structure) actively involved in
E,coli.
? Site-specific recombinations occur between DNA
molecules (or regions) having specific sequences
and is catalyzed by specific recombinases.
? Transposable genetic elements,found in all types of
organisms,jumps from one chromosomal location to
another via either direct transposition or replicative
transposition.
