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DNA Replication
Topoisomerase
• It wraps around DNA and makes a cut
permitting the helix to spin. Once DNA is
relaxed, topoisomerase reconnects broken
strand. Topoisomerase (type I &type II) is
an isomerase enzyme
Helicases
• Are a class of enzymes vital to all living
organisms. They are motor proteins that
move directionally along a nucleic acid
phosphodiester backbone, separating two
annealed nucleic acid strands (i.e. DNA,
RNA, or RNA-DNA hybrid) using energy
derived from nucleotide hydrolysis.
Primase
• A primer is a short RNA segment that is
complementary to a DNA segment, and is
necessary to begin DNA replication.
• Primase is of key importance in DNA
replication because no known DNA
polymerases can initiate the synthesis of a
DNA strand without initial RNA primers.
leading strand
• is the DNA strand at the opposite side of
the replication fork from the lagging strand.
It goes from a 5` - 3` direction (these
numbers indicate the position of the
molecule in respect to the carbon atoms it
contains).
lagging strand
• In DNA replication, the lagging strand is the DNA
strand at the opposite side of the replication fork
from the leading strand. It goes from 3' to 5' (these
numbers indicate the position of the molecule in
respect to the carbon atoms it contains).
• On the lagging strand, primase "reads" the DNA and
adds RNA to it in short bursts. Pol III lengthens the
bursts, forming Okazaki fragments. Pol I then
"reads" the fragments, removes the RNA using its
flap endonuclease domain, and adds its own
nucleotides (this is necessary because RNA and
DNA use slightly different kinds of nucleotides). DNA
ligase joins the fragments together.
Okazaki fragment
• When the lagging strand is being
replicated on the original strand, the 5'-3'
pattern must be used; thus a small
discontinuity occurs and an Okazaki
Fragment forms. These fragments are
processed by the replication machinery to
produce a continuous strand of DNA and
hence a complete daughter DNA helix.
DNA & RNA
Polymerases
• A polymerase is an enzyme whose
central function is associated with
polymers of nucleic acids such as RNA
and DNA. The most well-known function of
a polymerase is the catalysis of production
of new DNA or RNA from an existing DNA
or RNA template in the processes of
replication and transcription
DNA polymerase
• A DNA polymerase is an enzyme that assists in
DNA replication. Such enzymes catalyze the
polymerization of deoxyribonucleotides
alongside a DNA strand, which they "read" and
use as a template. The newly-polymerized
molecule is complementary to the template
strand and identical to the template's partner
strand.
• DNA-Polymerase initiates DNA replication by
binding to a piece of single-stranded DNA.
DNA polymerase
• Prokaryotic DNA
polymerases
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•
•
•
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• Eukaryotic DNA polymerases
• Pol α: acts as a primase
(synthesizing a RNA primer), and
then as a DNA Pol elongating that
(5 pol.)
primer with DNA nucleotides.
Pol I: implicated in DNA
• Pol β: is implicated in repairing
repair
DNA.
Pol II: involved in replication
• Pol γ: replicates mitochondrial DNA.
of damaged DNA
Pol III: the main polymerase • Pol δ: is the main polymerase in
eukaryotes
in bacteria
• Pol ε: uncertain.
Pol IV& Pol V: participates
in bypassing DNA damage.
• η, ι, κ, and ζ involved in the bypass
of DNA damage.
• There are also other eukaryotic
polymerases known, which are not
as well characterized: θ, λ, φ, σ, and
μ.
RNA polymerase
• RNA polymerase (RNAP or RNApol) is an
enzyme that makes an RNA copy of a DNA or
RNA template. In cells, RNAP is needed for
constructing RNA chains from DNA genes, a
process called transcription. RNA polymerase
enzymes are essential to life and are found in all
organisms and many viruses. In chemical terms,
RNAP is a nucleotidyl transferase that
polymerizes ribonucleotides at the 3' end of an
RNA transcript.
RNA polymerase
• Prokaryotic RNA polymerase
• α2: the two α subunits
assemble the enzyme and
recognize regulatory factors.
• β: this has the polymerase
activity (catalyzes the
synthesis of RNA) which
includes chain initiation and
elongation.
• β': binds to DNA
(nonspecifically).
• ω: restores denatured RNA
polymerase to its functional
form in vitro.
• Eukaryotic RNA polymerase
• RNAp I synthesizes a prerRNA , which matures into
rRNA which will form the
major RNA sections of the
ribosome.
• RNAp II synthesizes
precursors of mRNA
• RNA p III synthesizes tRNAs,
rRNA and other small RNAs
found in the nucleus and
cytosol.
Wobble base pair
• A wobble base pair is a G-U and I-U / I-A / I-C pair
fundamental in RNA secondary structure. Its
thermodynamic stability is comparable to that of the
Watson-Crick base pair. Wobble base pairs are
critical for the proper translation of the genetic code.
The genetic code makes up for disparities in the
number of amino acids (20) for codons (64), by using
modified base pairs in the first base of the anticodon. One important modified base is inosine
which can pair with three bases: uracil, adenine, and
cytosine.
• Another critical base pair is the G-U base pair, which
allows uracil to pair with two bases: guanine and
adenine.
Wobble base pairs for Uracil
Wobble base pairs for inosine
DNA Cloning
• A plasmid is a DNA molecule separate from the
chromosomal DNA and capable of autonomous
replication. It is typically circular and doublestranded. It usually occurs in bacteria,
sometimes in eukaryotic organisms. Size of
plasmids varies from 1 to over 400 kilobase
pairs (kbp). There may be one copy, for large
plasmids, to hundreds of copies of the same
plasmid in a single cell, or even thousands of
copies, for certain artificial plasmids selected for
high copy number. Plasmids can be part of the
mobilome, since they are often associated with
conjugation, a mechanism of horizontal gene
transfer.
• A cosmid, is a type of plasmid , constructed by the insertion of
cos sequences,
• Cos sequences are single stranded sequences of DNA, which
have been split from the parent molecule by a specific
restriction enzyme in such a way that the ends have specific
affinity for each other, and hence are known as cohesive ends.
• Cosmids are packaged in phage structures consisting of
proteins, which allows the foreign genes to be inserted into the
bacteria using transduction. If the Cosmids contain, for
example, genes for resistance against antibiotics, the
transfected bacteria are then able to survive and to spawn in a
nutrient solution containing the antibiotic and can thus be
selected. Cosmids can be used to build genomic libraries.