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Chapter 7
Genome and DNA Replication
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Introduction
A genome is all the genetic information that
defines an organism.
Microbial genomes consist
of one (usually) or more
DNA chromosomes.
This chapter explores the
structure of genomes
and their replication.
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Life is specified by genomes. Every organism,
including humans, has a genome that contains
all of the biological instructions needed to build
and maintain a living example of that organism.
The biological information contained in a
genome is encoded in its DNA and is divided
into discrete units called genes. Genes code
for proteins, rRNA, tRNA or small RNAs
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In 1909, Danish botanist Wilhelm Johanssen
coined the word gene for the hereditary unit
found on a chromosome. Nearly 50 years earlier,
Gregor Mendel had characterized hereditary units
as factors— observable differences that were
passed from parent to offspring. Today we know
that a single gene (or set of genes) provides the
complete instructions to make a functional
product, called a protein. Genes instruct each cell
type— such as skin, brain, and liver—to make
discrete sets of proteins at just the right times,
and it is through this specificity that unique
organisms arise.
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DNA: The Genetic Material
Two types of gene transfer are known:
- Vertical transmission: From parent to child
- Horizontal transmission: Bacteria seem to
operate differently where transfer of small pieces of
DNA from one cell to another cell of same species
or different species occurred frequently.
Fred Griffith’s work on horizontal gene transfer,
transformation led to the discovery that DNA is the
genetic material.
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Transformation
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Figure 8.24
In 1944, Avery, Macleod, and McCarty
discovered that the component
responsible for transforming harmless
strains of Steptococcus to a virulent
strain was DNA.
These results provided one of the
conclusive indications that DNA was
indeed the carrier of genetic material
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Genome organization
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Types of genes
Types of genes: regulatory and Structural
(transcribed or functional)
Structural gene: produces a functional RNA
(tRNA,rRNA, mRNA, small RNAs). Only
mRNA encodes a protein.
Regulatory gene: regulates the expression of
a structural gene.
- Does not encode an RNA
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Functional Units of Genes
A gene can operate independently of others.
- Or, it may exist in tandem with other genes
in a unit called an operon.
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DNA Structure
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Nucleosides and Nucleotides
DNA is a polymer of nucleotides.
Each nucleotide consists of three parts:
- Nitrogenous base
- Purine: Adenine (A) and guanine (G)
- Pyrimidine: Cytosine (C) and thymine (T)
- Deoxyribose sugar
- Phosphate
Nucleotides are connected to each other by
5´-3´ phosphodiester bonds.
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DNA structure elements
Bases in DNA:
Adenine (A), Guanine (G), Cytosine (C) and
Thymine (T)
A Nucleoside:
A base + Deoxyribose (a sugar)
A Nucleotide:
A base + Deoxyribose + Phosphate
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Organelle DNA
Not all genetic information is found in nuclear
DNA. Both plants and animals have an
organelle—a "little organ" within the cell—
called the mitochondrion. Each
mitochondrion has its own set of genes.
Plants also have a second organelle, the
chloroplast, which also has its own DNA.
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DNA Structure
Hydrogen bonding allows complementary
base interactions.
- A pairs only with T (via two H bonds).
- G pairs only with C (via three H bonds).
These interactions allow the two
phosphodiester backbones to come
together in an antiparallel fashion.
- Thus forming the double helix
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5’
3’
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DNA Structure
The DNA double helix
has grooves: a wide
major groove and a
narrow minor
groove.
- These provide DNAbinding proteins
access to base
sequences.
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RNA Structure
RNA differs from DNA:
- Usually single-stranded
- Contains ribose sugar
- Uracil replaces thymine
Figure 7.4B
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The Bacterial Nucleoid
Bacteria pack their DNA into a series of loops or
domains, collectively called the nucleoid.
- Loops are anchored by histone-like proteins
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The Bacterial Nucleoid
Figure 7.9
But how does DNA
achieve this
supercoiled state?
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DNA Supercoiling
Positive supercoils: DNA is overwound.
Negative supercoils: DNA is underwound.
Eukaryotes, bacteria, and most archaea
possess negatively supercoiled DNA.
Archaea living in acid at high temperature
possess positively supercoiled DNA.
Enzymes that change DNA supercoiling are
called topoisomerases.
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Supercoiling induced by separating the
strands of a helical structure. 'Iwist two
linear strands of rubber band into a righthanded doublehelix as shown. Fix the left
end by having a friend hold onto it. If the
two strands are pulled apart at the right
end, the resulting strain will produce
supercoiling as shown.
Removal of one turn induces structural
strain that can be accommodated by (c)
strand separation over 10.5 base pairs or
by (d) formation of a supercoil.
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Topoisomerases
Type I topoisomerases
- Usually single proteins
- Cleave one strand of DNA
- Relieve or unwind super coil
Type II topoisomerases
- Have multiple subunits
- Cleave both strands of DNA
- Introduce additional turns or coils
- Example: DNA gyrase
- Targeted by quinolone antibiotics
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DNA Replication
Extraordinarily important and complex
process. In bacteria, replication occurs at
750-1000 base-pairs/ second.
E.Coli makes errors with a frequency of
10-9- 10-10 base-pair replicated.
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Genetic Information flow
DNA Replication
Information Flows from DNA to RNA
to Proteins in 3 processes
Replication (DNA  2
DNAs)
2. Transcription (DNA mRNA,
rRNA, or tRNA
3. Translation (mRNA Protein)
1.
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Central dogma of molecular
biology
One way transfer of information
from nucleic acid to protein is
universal and holds for all forms of
life on the planet
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DNA Replication
Replication of cellular DNA in most cases is
semiconservative.
- Each daughter cell receives one parental
and one newly synthesized strand.
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DNA Replication
process
Replication in bacteria begins
at a single origin (oriC).
After initiation, a replication bubble forms.
-DNA methylation controls the timing of replication
- Contains two replication forks that move in opposite
directions around the chromosome
Replication ends at defined termination (ter) sites
located opposite to the origin.
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Replication Fork
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Replication Machinery (replisome)
The major proteins involved in DNA replication
include:
- DnaA: Initiator protein
- DnaB: Helicase
- DNA primase: Synthesis of RNA primer
- DNA pol III: Major replication enzyme
- DNA pol I: Replaces RNA primer with DNA
- DNA gyrase: Relieves DNA supercoiling
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Initiation of Replication
The start of DNA replication is precisely timed
and linked to the ratio of DNA to cell mass.
In E. coli, DnaA accumulates during growth,
and then triggers the initiation of replication.
- DnaA-ATP complexes bind to 9-bp repeats
upstream of the origin.
- This binding causes DNA to loop in
preparation for being melted open by the
helicase (DNaB).
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Figure 7.15
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Elongation of Replication
After initiation, each replication fork contains
two strands:
- A leading strand, which is replicated
continuously in the 5´-to-3´ direction
- A lagging strand, which is replicated
discontinuously in stages, each producing
an Okazaki fragment
- These are progressively stitched together to
make a continuous unbroken strand.
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Replisome
The cell coordinates the activity of two DNA
pol III enzymes in one complex.
- These two enzymes, together with DNA
primase and helicase, form the replisome.
The replisome ensures that the leading and
lagging strands are synthesized
simultaneously in the 5´-to-3´ direction.
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Figure 7.18b
Figure 7.18a
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Elongation of Replication
To remove RNA primers, cells use RNase H.
A DNA pol I enzyme then synthesizes a DNA
patch using the 3´ OH end of the
preexisting DNA fragment as a priming site.
Finally, DNA ligase repairs the
phosphodiester nick using energy from
NAD (in bacteria) or ATP (in eukaryotes).
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Figure 7.19
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Termination of Replication
There are as many as 10 terminator
sequences (ter) on the E. coli chromosome.
A protein called Tus (terminus utilization
substance) binds to these sequences and
acts as a counter-helicase.
Ringed catenanes formed at the completion
of replication are separated by
topoisomerase IV and the proteins XerC
and XerD.
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Plasmids
Plasmids are
extragenomic DNA
molecules.
- Much smaller than
the chromosome
- Usually circular
- Need host proteins
to replicate
Figure 7.23
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Plasmids
Plasmids can replicate in two different ways:
- Bidirectional replication
- Starts at a single origin and occurs in
two directions simultaneously
- Rolling-circle replication:
- Starts at a single origin and moves in
only one direction
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Figure 7.24
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Plasmids
Plasmids have tricks to ensure their inheritance:
- Low-copy-number plasmids segregate equally to
daughter cells.
- High-copy-number plasmids segregate randomly
to daughter cells.
Plasmids are advantageous under certain
conditions:
- Resistance to antibiotics and toxic metals
- Pathogenesis
- Symbiosis
Plasmids can also be transferred between cells.
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Analysis of DNA
Figure 7.27A
Restriction
endonucleases
cleave DNA at specific
recognition sites, which
are usually 4 to 6 bp
and palindromes.
- May generate blunt or
staggered ends
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Analysis of DNA
Agarose gel
electrophoresis
can be used to
analyze the DNA
fragments obtained
by treatment with
restriction
enzymes.
Figure 7.27B
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Analysis of DNA
The polymerase chain reaction (PCR) can
produce over a million-fold amplification of
target DNA within a few hours.
Figure 7.29
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