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Bacterial Chromosome Replication,
Structure and Segregation
Francis Crick and James Watson
Bacterial Chromosome Replication,
Structure and Segregation
Objectives
1. Develop a thorough understanding of DNA structure
because it is fundamental to the comprehension of:
a. gene replication
b. gene expression
c. gene regulation
d. gene mutation and repair of DNA damage
e. recombination
f. genome packaging and transmission
g. gene manipulation
h. genomics
2. Develop a thorough understanding of the process of
DNA replication.
3. Begin to comprehend the practical applications that
have been derived from the basic knowledge of
DNA structure and replication.
Experiments that demonstrated
that DNA is the genetic material
of bacteria and at least some
bacteriophage
or
Insights gained from transformation and
virus infection
The Griffith experiments
Heat-killed
pathogenic
encapsulated
bacteria can convert
nonpathogenic
noncapsulated
bacteria to the
pathogenic
capsulated form.
R indicates roughcolony formers.
S indicates smoothcolony formers.
Roman numerals
indicate serotype of
the capsid
polysaccharide, a
genetically
determined trait.
Bacterial type
Effect on mouse
A
Bacteria
recovered
None
Live type IIR
Nonpathogenic
B
Type IIIS
Live type IIIS
Pathogenic
C
Heat-killed
type IIIS
None
Nonpathogenic
D
Mixture of
live type IIR and
heat-killed type IIIS
Type IIIS
Pathogenic
Figure 6.1
Streptococcus pneumoniae colonies on
medium solidified with agar
R strain
S strain
Colonies of rough (R, small colonies) and smooth (S, large colonies)
strains of Streptococcus pneumoniae. The S colonies are larger because
each cell has a very thick, gelatinous, polysaccharide capsule, which is
missing in R mutants.
Photograph reproduced from from O. T. Avery, C. M. MacLeod, and M. McCarthy. 1944. J. Experimental Medicine 79: 137.
Avery, McLeod and McCarthy Experiment I
Heat
kill
Type IIIS
Type IIR
Type IIIS
Extract macromolecular components
Polysaccharides
Lipids
Proteins
Nucleic acids
(DNA + RNA)
TRANSFORM LIVE TYPE IIR CELLS
?
IIR
IIR
IIR
IIR + IIIS
The transforming principle is a nucleic
acid, NOT a polysaccharide
Avery, McLeod and McCarthy Experiment II
Type IIR
Type IIIS
Fig 2.3 in Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
DNA, rather than RNA, is the D. pneumoniae transforming principle
Hershey-Chase experiment demonstrating that DNA is
the genetic material of bacteriophage T2
Alfred Hershey and Martha
Chase knew that T-even
bacteriophage (T2, T4, T6)
consist of protein and DNA.
They also knew that phage
proteins contain sulfur and
no phosphate, and phage
DNA contains phosphate but
no sulfur. They decided that
these characteristics could
be used to determine
experimentally if the genes
of bacteriophages consist of
protein or DNA.
Hershey-Chase experiment demonstrating that DNA is the genetic
material of bacteriophage T2
Hershey and Chase also knew that many progeny phage are produced and
released after a phage infected a bacterial cell.
NEXT
Hershey-Chase experiment demonstrating that DNA is
the genetic material of bacteriophage T2
Armed with all this knowledge, Hershey and Chase designed an
experiment to determine if protein or DNA is the “genetic material”,
i.e. the substance that is passed from one generation to the next.
A. Preparation of the experimental material: radioactively-labeled T2
bacteriophage.
NEXT
Modified from Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing s Benjamin Cummings.
B. Part of the Hershey and Chase experiment that showed that
DNA is the genetic material of bacteriophage T2.
Collect cells by centifugation to
remove phage "ghosts"
32P
35
S
EUREKA!
DNA is transmitted from the parental phage to the progeny
phage and protein is not!
The secret is in the molecular structure of DNA!
Erwin Chargaff did quantitative chemical
analyses of of DNA from different organisms and
observed that:
[A] = [T] and [G] = [C], but [A+T]
≠ [G+C]
"Pairing I used later, translating my word into what had become a
slogan. I did not say they were in a double structure, no. That is Crick
and Watson. The helix is a gimcrack. The fact that it is double is
Erwin
important because it is an automatic way of reproduction. I never
Chargaff
claimed it was my idea, and I don't wish to."
Interview with Erwin Chargaff, OMNI, 7, no. 9 (June 1985): 132. June 1985.
Rosalind Franklin
Maurice Wilkins
Concluded from X-ray diffraction data that
DNA is a helix
DNA X-ray diffraction pattern
DNA
A double helix with paired bases: potentially contains information
for its own replication (base pairing) and can encode gene
products such as enzymes (base sequence).
James Watson and Francis Crick
Used the data to develop a structure that fit the data
DNA
2 nm (20 Å)
3’
5’
Minor groove
Major groove
2.2 nm
Major groove
5’
3’
Hydrogen bonds
Deoxyribose-phosphate Backbones
1 turn = 3.4 nm = 10 base pairs
Minor groove
1.2 nm
Deoxyribose-phosphate Backbones
Modified from Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
A
B
DNA Replication
In 1953 Watson and Crick
PREDICTED that DNA
replication is semiconservative, meaning
that each strand can act
My Heroes
as a template for the
synthesis of its
complementary strand: two new copies
are made, each consisting of an “old”
(template) strand and a “new” strand.
3’
5’
A
B
In 1958, Matthew Messelson and
Franklin Stahl confirmed experimentally
that DNA replication is semiconservative.
3’
5’
3’
5’
The Messelson Stahl Experiment
In 1958, Matt Messelson and Frank
Stahl designed an experiment to
discriminate among these
possibilities. Important features:
1. E. coli could grow in a medium
having 15NH4Cl instead of the
normal ammonium chloride. The
heavy nitrogen eventually replaced
the normal nitrogen in the cell’s
molecules. If returned to the
normal medium, the 14N isotope
would subsequently be
incorporated.
2. DNAs having the heavy or light
isotopes of N have a different
density and can be separated in a
CsCl buoyant density gradient.
14N
Hybrid
15N
1:1
3 : 1
The Messelson Stahl Experiment
Question
What would Messelson and Stahl have observed in the
CsCl equilibrium gradients after one and two
generations if DNA replication would have been
A. Conservative: the parental strands remain together
and the two daughter strands together are a replica
of the parental dsDNA?
B. Dispersive: both strands of the parental DNA are
broken up and pieces, together with additional
nucleotides, are assembled into two DNA molecules
in which both strands have some parental and some
new pieces? The pieces could be individual
nucleotides.
Components of nucleotides
Fig.1.2
BASES
Purines
Purine
Adenine
Guanine
Pyrimidines
Pyrimidine
Cytosine
Uracil
Thymine
O
Sugars
(Pentoses)
Na+ −O P O− +H
O−
K+
2-Deoxyribose
Ribose
Phosphate
Nucleotide and nucleosides
DNA nucleotide
dAMP
RNA nucleotide
UMP
Nucleotides
5’
5’
5’ dGMP
3’
3’
3’ dCMP
9
5’
3’
Fig.1.2
5’ dGTP
Phosphodiester bonds and polarity of polynucleotides
5’ end
3’ end
DIRECTION OF NUCLEOTIDE
SEQUENCE
3’
5’
upstream
downstream
site
Oligonucleotides < 100 bases
Polynucleotides > 100 bases
Fig 1.3
Base pairing with
hydrogen bonding
Fig 1.4
Polarity and Antiparallel Orientation of Strands in DNA
Bases can be flipped out of the double helix by DNA
processing and repair enzymes.
Antiparallel strands
Base flipped
DNA Base Flipping by Enzymes
DNA modifying
enzymes
N-glycosylases
(remove bases
from pentose in
DNA backbone)
DNA repair
endonucleases
Model of DNA Double Helix Showing Potential Protein
Contact Points the Major and Minor Grooves
Phosphate-ribose backbones
Proteins interact with DNA mainly by
binding to atoms of bases exposed in the
major groove (shown in red).
Minor
groove
Major
groove
Modified from Fig 7.5 in Madigan and Martinko 2006. Brock Biology of Microorganisms.
Architecture of Prokaryotic Chromosomes
1. Most of the prokaryotes have a single circular chromosome.
2. Some prokaryotes have two or three, perhaps even more,
circular chromosomes.
3. Some prokaryotes have at least one linear chromosome of
either the “invertron” or “hairpin telomere” type.
4. Organisms that have circular chromosomes tend to have
circular plasmids, and organisms that have linear
chromosomes tend to have linear plasmids. However, this is
not a hard and fast rule: there can be disagreements! For
example, Rhodococcus fasciens has a circular chromosome,
but at least one of its multiple plasmids is linear.
5. The shape of the chromosome affects several aspects of
chromosome replication and distribution.
Some examples of bacterial genome organization
Bacterium
Escherichia coli K12
Bacillus subtilis
Xylella fastidiosa
Chromosome(s)
one circular (4.6 Mb)
one circular (4.2 Mb)
one circular (2.7 Mb)
Brucella melitensis
Vibrio cholera
Deinococcus radiodurans
Rhizobacterium melliloti
two
two
two
two
Paracoccus denitrificans
three circular (2.0 + 1.1 +
0.64 Mb)
Streptomyces coelicolor
one linear (8.7-Mb
invertron)
one linear (356 Kb) + one
circular (31 Kb)
Borellia burgdorferi
one linear (0.9-Mb hairpin)
multiple linear and circular
(9 - 54 Kb)
circular
circular
circular
circular
(2.1
(2.9
(2.6
(3.4
+ 1.2 Mb)
+1.1 Mb)
+ 0.4 Mb)
+ 1.7 Mb)
Plasmids
two circular (51 + 1.3 Kb)
two circular (177 + 45 Kb)
one circular megaplasmid
(1.4 Mb)
Expanded from Ochman H. 2002. Bacterial evolution: chromosome arithmetic and geometry. Curr. Biol. 12:R427–28.
Replication of Circular Chromosomes
The E. coli Paradigm
Proteins involved in E. coli DNA replication
Protein
Gene
Function
DnaA
dnaA
Initiator protein, primosome (priming complex) formation
DnaB
dnaB
DNA helicase (strand separation; hexamer)
DnaC
dnaC
Delivers DnaB to replication complex
SSB
Primase
ssb
dnaG
Binding to single-stranded DNA
RNA primer synthesis
DNA Pol III
11 genes DNA replication (presented in next slide)
DNA Pol I
polA
Primer removal, gap filling
DNA ligase
lig
Sealing DNA nicks
DNA gyrase
α subunit
gyrA
DNA supercoiling (unwinding)
Nick closing
β subunit
gyrB
ATPase
topA
DNA supercoiling (DNA “packaging”)
Topo I
Pol III
TABLE 1.1
The 11 polypeptides of the E. coli DNA Pol III
(holoenzyme)
Subunit
Gene
Function
α
ε
RNase H
dnaE
dnaQ
rnhA
θ
β
τa
holE
dnaN
dnaX
γb
δ
δ’
χ
ψ
dnaX
holA
holB
holC
holD
Polymerization (catalytic subunit).
3’ → 5’ editing exonuclease.
Removes RNA primers? Endonuclease that
cleaves RNA in RNA/DNA duplexes. Primer
removal in eukaryotes.
Present in core.
Sliding clamp.
Organizes complex; joins leading and lagging DNA PolIII
complexes.
Clamp loading.
Clamp loading.
Clamp loading.
Clamp loading.
TABLE 1.1
Clamp loading.
a
b
Full-length translation product of dnaX gene
Shorter product of dnaX gene produced by translational frameshifting (Chapter 2)
Some Properties of DNA Polymerases
1. All DNA polymerases require a template and a
PRIMER (3’-OH end). Primers can be DNA or RNA.
2. In addition to the ability to replicate DNA in the
5'→3' direction, the DNA polymerases have a 3'→5'
exonuclease activity (for proof-reading).
3. Pol I & II also have a 5'→3' exonuclease activity
for the degradation of primers or widening of gaps
during DNA repair (Pol III may lack this activity).
IMPORTANT NOTE: RNA polymerases do NOT require
a primer for the initiation of RNA synthesis!
Primases are RNA polymerases.
OH
Functions of the primer
and template during
DNA synthesis
Fig. 1.7
OH
C
The Basics of DNA Replication
1. Replication starts at origins of replication (ori sites).
2. Once initiated, replication proceeds bidirectionally on
both template strands by leading- and laggingstrand synthesis.
3. Leading-strand synthesis is continuous (5’ Æ 3’).
4. Lagging-strand is discontinuous and involves the
synthesis and joining of many “Okazaki” fragments.
All Okazaki fragments are synthesized in the same
orientation (5’ Æ 3’).
5. Replication ends when replication complexes collide.
However the regions in the genomes where collisions
occur may be determined by replication-termination
(ter ) sites.
DNA Replication: Initiation
1. DNA replication starts at origins of replication (ori )
AT-rich
12-bp
3 AT-rich
13-mers
4 DnaA
recognition sites
Fig 1.16
The ori-C site of E. coli is slightly less than 260 bp long and
includes 4 DnaA boxes, 3 AT-rich 13-mers, a 12-bp AT-rich strandseparation region, and 11 GATC/CTAG Dam methylation sites.
Dam = deoxyadenosine methyl transferase
What is a “site”? There are sites within sites!
What is a “box”?
DNA Replication: Initiation
oriC
10 to 12 molecules of DnaA protein
with ATP bind to the DnaA boxes of oriC
and form a complex that winds DNA
around itself and causes opening of the
helix in the adjacent AT-rich regions.
3’
5’
DnaC protein
delivers and
clamps DnaB
helicase
protein as a
hexamer to
each strand in
the open DNA
3’
“bubble”.
5’
3’
DnaG primase
synthesizes primers
initiating replication.
3’
5’
5’
3’
5’
Primosome
SSB
5’
3’
Helicase begins unwinding more
DNA by sliding along each strand in
the 5’ Æ 3’ direction. Strands are
kept separate by binding of single
strand binding protein (SSB).
Bidirectional Replication
2. Replication starts at origins of replication and proceeds
bidirectionally along both strands of the helix until one
replication complex collides with another going in the opposite
direction.
θ (theta) DNA
structure
DNA Replication: Elongation
Model for leading and lagging strand replication
GTC – sites for the initiation of RNA primer synthesis: 4 sites shown
DNA gyrase (a type II
topoisomerase)
Dna B
helicase
DNA Pol III
DIMER
Primer RNA
DNA helicase
unwinding DNA
MORE
3’
5’
DNA Replication: Elongation
Model for leading and lagging strand replication
Okazaki
fragment
~1000 b
DNA G primase makes
new RNA primer
Primer degraded and
gap filled by DNA Pol I
LINK
Videos
Organization and dynamics of the E. coli replisome
helicase
primase
clamp
Modified from Fig. 4 Johnson and O'Donnell
(2005). Annu. Rev. Biochem. 74:283-315.
Completion of
Lagging Strand
Synthesis
A. DNA Pol III starts from a
primer and replicates
until it reaches the
primer of the previous
Okazaki fragment.
B. DNA Pol III is released
from lagging strand and
“snaps back” to nearest
unused primer to start
new Okazaki fragment.
C. DNA polymerase I
degrades primer between
Okazaki fragments in 5’
to 3’ direction and fills
gap with DNA.
D. DNA ligase seals last gap.
DNA polymerase III
DNA polymerase I
DNA ligase
ATP
ADP + Pi
A Summary of
DNA Replication
Termination of Replication
5. Termination of replication occurs when replication complexes
collide. At least some prokaryotes have termination sites (ter ),
which are inverted repeats of many short repeats of nucleotide
sequences that bind proteins and progressively slow down the
movement of replication forks (replication complexes). The ter
sites are not essential: deletion of these sites from the Bacillus
subtilis chromosome slows cell growth but does not block cell
division. Eukaryotes and viruses may not have ter sites.
Fig 1.18
Stalling of fL
Tus (E. coli ), RTP (B. subtilis) are replicationtermination proteins that bind to ter sites
Stalling of fR
Replication of Linear Chromosomes: the end problem
Why is there no primer to
synthesize DNA at the
ends of chromosomes?
There is no template for
the primase to make a
primer!
Why can't DNA polymerase
replace the RNA primer?
It can not synthesize DNA in
the 3’ 5’ direction and it
can not initiate replication in
the 5’ 3’ direction unless it
is primed!
Replication of Linear Chromosomes
Some solutions to the end problem
1. The linear chromosomes of some bacteriophages have singlestranded cohesive ends and are circularized after entry into the
host cell.
Example: The ~48,000-bp phage λ chromosome.
cos sequence
cos
cos
Replication of Linear Chromosomes
The ends of the linear chromosomes
and plasmids of some organisms are
covalently-closed hairpin structures.
TIR
Borrelia burgdorferi
TIR
African swine fever virus
Vaccinia virus
L ~25-bp inverted repeat R
Replication initiated
from internal origins
Replication of Linear
Chromosomes with
Hairpin Ends
Replication produces a
circular dimer
“Telomere resolvase”
recognition site
Telomere resolvase cleaves
and ligates appropriate
ends to form hairpins
Replication of Linear Chromosomes: Invertrons
ITR
5’ TP
(Terminal
protein)
tp
pol
3’
Terminal Protein
O
OH
Ser
Thr
or Tyr
Replication of the ends of linear
chromosomes of the invertron type is
primed by a nucleotide linked to a protein
(terminal protein = TP). The TP remains
permanently attached to the 5’ termini.
Examples: Streptomyces, B. subtilis Φ29
3’
Replication of Linear Chromosomes
Some solutions to the end problem
4. Eukaryotes solve the endreplication dilemma by
extending their 3’ end by
means of of a reverse
transcriptase that has its own
RNA template: telomerase.
Second-strand synthesis require
primase and DNA polymerase
Replication
RNA Primer
Some telomeres are 100 to
200 Kb long
Replication Error Editing
and
Methyl-directed Repair
Nick Translation
A
5’Æ3’ exonuclease
3’
5’
RNA
5’
3’
B
Fig 1.11
DNA polymerase I can remove
the nucleotides of an RNA
primer (or nicked DNA strand)
and simultaneously fill the gap
with a new DNA strand by virtue
of its “nick-translation” activity.
C
5’Æ3’ exonuclease
Pol
Exo
Nick translation
Mistakes in base pairing can lead to changes in
the nucleotide sequence of DNA: mutations
Fig
1.13
GMT mismatch
During replication
(unedited)
dsDNA with
One mismatched
base pair
Replication
Wild
type
Mutant
Proofreading and Editing
A mismatch in pairing at the terminal base pair causes the DNA
polymerase to pause: the last base in the new strand is
removed by the 3’ Æ 5’ exonuclease activity of DnaQ, the ε
subunit of DNA polymerase III, before replication continues.
Proofreading
Removal of
mismatched
base
Error corrected
as synthesis is
resumed
Proofreading and Editing
DnaQ can remove only mismatched nucleotides that are
located at the 3’ end of the newly synthesized
strand. If the polymerase adds even one additional
nucleotide, then DNA Q no longer can remove the
mismatched base. This situation can result in a
mutation, unless the damage is repaired by a
different mechanism.
What is a mutation?
A mutation is a heritable change in the nucleotide
sequence of the genome of an organism.
As we will see later in this class, there are many lesions
in DNA molecules, most of which can be repaired.
Only a small fraction of DNA lesions actually result in
mutations.
If a mismatch escapes repair by replication editing, it still
can be fixed by methyl-directed DNA mismatch repair.
In E. coli, newly-synthesized DNA strands can be distinguished
from “old” template strands by delayed methylation of A in 5’GATC-3’ sites by deoxyadenosine methyltransferase (DAM).
CH3
5’
3’
GATC
CTAG
CH3
Both strands
methylated
Pol III
Pol III
3’ old
5’ new
3’ new
5’ old
Hemimethylated DNA
Post-replication base modifications are mechanisms involved in:
1. Methyl-directed DNA mismatch repair.
2. Protection of DNA from cleavage by cellular site-specific
restriction endonucleases.
3. Gene expression in some prokaryotes and all eukaryotes.
Methyl-directed mismatch repair
Dam
targets
Dam = deoxyadenosine methyl transferase
Cut?
MutG cuts at nearest
unmethylated GATC.
UvrD helicase unwinds
strands past mismatch.
New strand is degraded in
the 5’Æ3’ direction by
RecJ or ExoVII if nick is
on 5’ side, or in the 3’Æ5’
direction, or by ExoI or
ExoX if the nick is on the
3’ side of the mismatch.
C
T
Cut?
Dam adds
methyl to 6
position of A
DNA pol III
MutS, MutL and
MutG endonuclease
bind to mismatched
bp.
Chromosome Partitioning into
Daughter Cells
Replication and Partitioning
SeqA at hemimethylated GATC sites?
Fig 1.19
FtsZ ring
The binding of SeqA to hemimethylated GA*TC/CTAG sequences
protects against premature initiation of replication before the cell
cycle is complete.
SeqA
Visualization of
hemi-methylated
DNA at oriC by
binding of
SeqA-YFP protein
(detected by
yellow
fluorescence in UV
light by confocal
microscopy).
SeqA
probably is
NOT directly
involved in
binding of
oriC to the
membrane
Jellyfish have yellow and green
fluorescent proteins.
Fig 1.21
The polymerization of the FtsZ tubulin into a
ring determines the site of cell division and
is critical for the correct distribution of
daughter chromosomes during cell division
(cytokinesis).
FtsZ Ring
(FtsZ-GFP)
(Landing pad for recruitment of
other proteins to division site -GTPase, required for cytokinesis)
Modulators of FtsZ ring,
connecting the ring to the
cytoplasmic membrane
Coordination of chromosome
distribution with cell division, ATPdependent DNA translocase, binds
Topo IV (decatenase) and XerCD
(dif, dimer resolvase)
Peptidoglycan synthase
Peptidoglycan hydrolase
Escherichia coli, Bacillus subtilis and Caulobacter crescentus all
divide by assembling an FtsZ tubulin ring, but each species uses
different proteins and mechanisms to direct FtsZ to its proper
location. NOTE the polar proteins, which oscillate from pole to
pole in E. coli. Helical filaments formed inside the cell membrane
by MreB, an actin-like protein, are involved in the oscillation of the
Min proteins.
MreB and ParM are Actin Homologs
MreB is always encoded by a gene on the bacterial chromosome
and controls a wide variety of cellular functions, including:
1. Cell shape (mutants form spherical rather than elongated cells).
2. Localization of polar proteins (proteins are mislocalized in
mutants)
3. Chromosome segregation (chromosomes are not separated into
daughter cells in mutants).
MreB self assembles into filaments in vitro.
In vivo, MreB forms bundles of filaments that are arranged in a
helical structure inside the cell. Like the actin filaments of
eukaryotic cells, MreB filaments appear to assemble and
disassemble from opposite poles (ends).
The ParM actin-like protein always is encoded by genes located on
plasmids and is dedicated to the proper partitioning of plasmid
DNAs during cell division.
What have we learned from the partitioning of
Plasmids?
Many plasmids have partitioning systems that prevent curing by
random loss.
Single-copy plasmids such as P1, F, and R1 have partitioning
systems that consist of the following components:
1. A cis-acting “centromere-like” binding site on the plasmid DNA,
parC.
2. Two proteins, the actin-like ParM and a parC-binding protein,
ParR.
Multiple copies of ParR, or its equivalent in other plasmids, bind to
parC, a centromere-like sequence found in single-copy
plasmids, forming the equivalent of a “kinetochore”. The
parC/ParR complex then promotes polymerization of ParM into
filaments between the two plasmids, pushing them from the
middle to the quarter positions of the dividing cell. Like actin,
ParM has ATPase activity.
R1 plasmid segregation
F plasmid segregation
SopA aster
Aster
oscillates
between
kinetochores
ParM
From Z. Gitai 2006. Curr. Biol. 16: R133-R136.
What does plasmid segregation tell us about
bacterial chromosome segregation?
A lot, but not the whole story...
1. Bacteria have several genes that code for homologs of the Par
proteins of plasmids, including MreB (many bacteria), called
Soj in Bacillus subtilis.
2. The MreB proteins of bacteria are actin homologs.
3. In Bacillus subtilis, filaments of a ParM-like protein, Soj, are
linked to a parC-like nucleotide sequence near ori through
multiple copies of a ParR-like protein, Spo0J.
4. Bacterial partitioning proteins also show “oscillatory cycles” in
vivo similar to the SopA asters of the F plasmid.
5. Electron cryotomographic examination of bacterial cells has
revealed exquisite internal compartmentalization, including a
cytoskeleton consisting of numerous bundles of filaments
similar to those of the cytoskeleton of eukaryotic cells.
Resolution of
Chromosome Dimers
and
Concatemers
dif
Chromosome dimers are
generated by homologous
recombination during replication
dif
dif
dif
Dimer
dif
dif
dif
dif
dif
dif
Chromosome dimers are
resolved by XerCD
resolvase through
site-specific recombination
at dif during cytokinesis.
Question for Thought
What happens if two circular DNA molecules, such as bacterial
chromosomes or plasmids, are joined by two, three or more
“crossovers” (recombination events) that have occurred at
different sites?
Can you develop a rule about the structures that are produced
by multiple recombination events?
Which of these structures would require XerCD site-specific
recombination at dif for their resolution during cytokinesis?
Concatemers of Chromosomes
are Resolved by type II
Topoisomerases (Topo IV in E.
coli )
DNA Packaging in Nucleoids
Electron micrograph of uranyl acetate/lead-citrate
stained thin section of an E.coli cell showing
electron-dense (clear) regions of condensed DNA
Nucleoid
Fig. 1.22
NOTE: The Planctomycetes are prokaryotes that have complex
internal membrane structures.
Members of
the genus
Gemmata have
nucleoids that
are enclosed
by an envelope
resembling
nuclear
membranes of
eukaryotes.
From J. A. Fuerst: Intracellular
Compartmentation in
Planctomycetes. Annu. Rev.
Microbiol. 2005. 59:299–328
Organization of Prokaryotic Chromosomes
Nucleoid
Plasmid DNA
DNA is tightly compressed into nucleoids
and spreads when cells are gently lysed.
Chromosome
Compacting of DNA into a Repetitive Stable
Structure by E. coli MukBEF Condensin
Hinge
Condensation by
sequential binding of
MukBEF
Arms
ABC Heads *
Relaxation by extension
and sequential release
of MukBEF
From: Case et al. Science 305: 222-227 (2004)
* ABC = ATP-binding cassette
Speculative Model of Condensed Prokaryotic Chromosome
~30 kb domains of
supercoiled DNA
protruding from between
heads of MukBEF dimers
Helically propagated
MukBEF complexes
From: Case et al. Science 305: 222-227 (2004)
Electron micrographs of bacterial nucleoids showing
supercoiled and partially-relaxed loops of DNA
Partially
relaxed
Supercoiled
Highly supercoiled
Supercoiled DNA
Nick
Break one
strand
Seal
Break one
strand
Relaxed
covalently closed
circular DNA
Relaxed nicked
circular DNA
Rotate one end
of broken strand
around helix and
seal
Proteins
Inside cells, DNA is highly supercoiled
associated with proteins.
MORE
Supercoiled
circular DNA
Supercoiled
domain
A
Supercoiling and
relaxation of helical
double-stranded DNA
Fig. 1.24
Enzymes are necessary to increase or decrease DNA
supercoiling: gyrases and topoisomerases
Topo I
Fig 1.25
Topo II and gyrase
Such enzymes are essential for
replication and DNA repair
Gyrase (Topo II)
Action
DNA
dimer
5’ PO4 ends linked to tyr122 of GyrA subunits.
From Costenaro, L., Grossmann, J.G., Ebel, C. and Maxwell, A. Small-angle X-ray scattering reveals the solution structure of
the full-length DNA gyrase a subunit., Structure 13: 287–296, 2005
Topo I
Topo II and gyrase
Summary of Supercoiling and Topoisomerases
Positive and negative supercoiling can occur in the same DNA
molecule due to the action of helicases, gyrases and
topoisomerases.
Double-stand “breaks” by Topo II to relax negative or positive
supercoiling or gyrase (+ ATP) to add negative supercoiling
Any helicase
Topo I single-strand nicks
relax + or − coiling
SV40
replisome
Coordination of Chromosome Replication
with the Rate of Cell Division
Cells grow and divide at different rates in different media.
For example, the doubling times for E. coli are:
~ 140 min in succinate minimal medium.
~ 70 min in glucose minimal medium.
~ 30 min in Luria broth.
The rate of DNA synthesis in E. coli is the same in all media:
approximately 230,000 nucleotides are polymerized by the 4
DNA pol III complexes involved in bidirectional replication.
The E. coli chromosome is a 4.63 Mbp circle.
It takes ~40 min to complete one round of DNA replication.
Therefore, rapidly dividing cells would lose chromosomes, and
slowly dividing cells would accumulate DNA, unless
chromosome replication is synchronized in some way with the
rate of cell division.
Coordination is accomplished by controlling the initiation of rounds
of replication.
Coordination of chromosome replication with generation time
I = 70 min
I = 30 min
Fig 1.20
Antibiotics that Block DNA Replication
(a quinolone)
Ciprofloxacin
Chemically synthesized
fluoroquinolone
GyrA of DNA gyrase
(also other Topo II)
WARNING!
The indiscriminate use of Cipro already has resulted in extensive
selection of bacterial pathogens that are resistant to
fluoroquinolone antibiotics, even though it was thought that it
was “virtually impossible” for microbes to acquire the multiple
mutations that were deemed necessary to develop this trait.
Fig 1.5
Synthesis of
deoxyribonucleotides
from ribonucleotides
Abbreviations:
THF, tetrahydrofolate
DHF, dihydrofolate
Inhibitors of DNA Synthesis
Ciprofloxacin
4-quinolone
Mitomycin C
Nalidixic acid
DNA remains linked to tyr122 of
GyrA subunits; gaps not closed.
Novobiocin (a coumarin)
Crosslings DNA strands Competitive inhibitor of ATPase activity of GyrB