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III
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Central Dogma
This is the flow of genetic information within a biological system
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RNA can be converted back to DNA by reverse transcription
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B
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This is because of base
pairing
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Each original strand of DNA functions as a template; the sequence of these nucleotides
in each strand is copied in order to form a new complementary strand
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This process produces two identical daughter duplexes from the original parental
double helix
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Features of Replication
a
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Each parental strand remains associated with its corresponding, newly
synthesised complement (green) so that each DNA duplex contains one
parental and one new daughter strand
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Origin of replication
The origin of replication is the site at which replication begins
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The origin of replication is known as the ori site

c
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Replication occurs 5’-3’
The parental strand is double stranded with one strand 5’-3’ and the second
strand 3’-5’ (DNA helix is anti-parallel)

The replication fork is moving left to right
The antisense strand acts as a template so that the daughter strand produced is
an exact copy of the sense strand
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The replication fork is moving right to left
The parent sense strand acts as a template so that the daughter strand
produced is an exact copy of the antisense strand
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e
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This disrupts hydrogen bonding between base pairs (that are holding the two
strands together)
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As a result, the enzyme
gyrase (which is a topoisomerase) will introduce negative supercoils (at the
expense of ATP) by breaking the phosphodiester bonds, thus introducing the
negative supercoil
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In addition, single stranded DNA binding proteins bind to the single strand
of DNA in order to prevent reannealing
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Replication is semidiscontinuous
The leading strand is copied continuously whereas the other parental strand
(known as the lagging strand) is copied intermittently or discontinuously
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Garrett, R
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M
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Biochemistry
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Belmont, CA: Brooks/Cole Cengage Learning, p
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2
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It requires all four
deoxynucleotides; a template and a primer (a single strand (ss)DNA or RNA (with a
free 3’OH) that is able to pair with the template to form short double stranded
regions

a
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coli DNA polymerase

E
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DNA Polymerase I
Polymerase I has three active sites and is its own “proof reader” and “editor”

ii
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coli chromosome, a DNA polymerase
III sites at each replication forks

b
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The exonuclease activity
As stated, the 3’-5’ exonuclease activity serves as a proofreading function –
removing bases that have been incorrectly matched so that the polymerase
can try again
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DNA polymerase III
DNA replication is carried out by this holoenzyme
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The two polymerases or “dimeric” polymerase – one subunit will synthesise
the leading strand while the other subunit will synthesis the lagging strand
The core enzyme consists of 3 subunits (α, ε, θ)
-

α is the polymerase subunit

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ε is the 3’-5’ exonuclease subunit

-

θ is the subunit that stabilises the ε subunit

The β subunit is a dimer that forms ring around the DNA molecule – forming a
clamp
Additional subunits (γ, δ, δ’, χ, ψ and τ) are responsible for adding and
anchoring the β unit core structure
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Mechanisms of replication in E
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The replisome
Consisting of DNA unwinding proteins; the priming complex or primosome
and DNA polymerase III holoenzyme which is comprised of two replicative
polymerases

i
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DNA-B (which is a helicase) is brought by DNA-C, and further
unwinds the DNA strands
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Elongation and Termination
Remaining supercoiling is relieved by topoisomerase II (a DNA gyrase)
Elongation involves DnaB (a helicase) which unwinds the strand and single
stranded binding proteins bind in order to keep the two strands
separated
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A protein known as ter protein is also involved
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b
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DNA
polymerase II unclamps and then reclamps on the lagging strand whenever the
primer of the okazaki fragments is encountered
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DNA replication is Eukaryotes
Eukaryotes contain at least 19 different DNA polymerases
a
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DNA pol α – contains 4 subunits with a processivity of 200 and is involved in
initiation of nuclear DNA replication
c
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coli but is more complex in that the human cell
contains 6 billion base pairs of DNA that needs to be copied and contains an origin
of replication every 3 to 300 kbp
C
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RNA functions as a single stranded template

this process makes
use of a tRNA primer (which binds and is the start of transcription)

by means of the
enzyme, reverse transcriptase, transcription occurs from the tRNA primer, 5’ – 3’
direction
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this produces a single
strand of DNA
Then DNA polymerase generates a complimentary strand of DNA producing double
stranded DNA called complimentary DNA or cDNA

D
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Genetic Recombination Introduction
Genetic recombination is the exchange or incorporation of a DNA sequence with or into
another DNA sequence
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Homologous recombination
This occurs between similar sequences and results in arrangement of genes into
new combinations (repair)
2
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Transposition
This is the insertion (by an enzyme, generally transposase) of a transposon which is
a mobile genetic element
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Strand invasion then occurs where the two duplexes unwind and the free single
strand end of the one duplex starts to base pair with a nearly complimentary single
stranded region along the intact (unwound) strand in the other duplex
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This intermediate is known as Holliday junction
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Two more nicks occur either in the same strand (this is known as patch recombination) as
before or the other strand (this is known as splice recombinants)

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