BIOL 200 Lecture Notes - Phosphodiester Bond, Provirus, Transfer Rna

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6 Apr 2012
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Naveen Sooknanan McGill Fall 2011
1
DNA Replication:
DNA needs to replicate before cell division in order to produce daughter strands which are DNA
duplexes identical to the parental DNA molecule.
DNA needs to replicate faithfully so as to not transfer mutations to RNA, and eventually,
to proteins
Mutations can alter protein shape altering their function or rendering them useless
altogether
In DNA replication, either parental DNA strand can be used as a template to synthesize a
corresponding daughter strand, producing two daughter strands in total which are identical to the
parental duplex
Matthew Meselson and Franklin Stahl discovered that DNA replicated in a semi-
conservative manner
In the conservative replication mechanism, the parental DNA molecule remains intact after
replication producing the original strand and a completely new daughter strand
The parental DNA duplex remains intact
In the semiconservative replication mechanism, the parental duplex splits up after the first
replication creating ½ new and ½ old duplexes
In the second replication, each of these strands is used as a template producing two
hybrid DNA duplexes and two completely new ones
Meselson and Stahl discovered that DNA replicated in a
semiconservative manner by conducting the following experiment
on an E. coli gene:
Parental DNA was synthesized using 15N (heavy nitrogen)
These parental DNA molecules were placed in a medium
containing 14N where it was allowed to replicate twice
o This assured that any daughter strands would be
lighter than parental DNA because they would be
made from 14N
o Upon centrifugation, parental DNA duplexes would produce
heavy bands, new DNA would produce a light band, and
hybrid DNA would produce a medium band
The centrifugated product after 1 replication showed only medium
bands, showing a semiconservative mechanism
Upon 2 replications, the centrifugated product showed medium and
light band, showing the presence of only hybrid and new strands
This shows that DNA replicates in a semiconservative manner
Nucleotide polymerization in replication requires DNA polymerase as was as deoxynucleoside
5’ triphosphates (dNTPs)
Replication also requires a primer which can be either DNA or RNA
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Naveen Sooknanan McGill Fall 2011
2
o Primers are usually RNA in vivo, whereas DNA primers are typically used in
vitro
o Contrasts RNA polymerization, which does not require a primer
Replication proceeds in the 5’ 3’ direction like in RNA polymerization
dNTPs are added one by one to the free 3’ hydroxyl end of the primer
The primer can be very short, only 5-100 nucleotides long
and it anneals to a specific sequence on the template strand
Every annealed dNTP releases 1 pyrophosphate molecule
The primer is removed after replication and replaced with
DNA
In order for replication to occur, the duplex must first be unwound. This is done by an enzyme
called DNA helicase.
Unwinding is initiated at specific points on the DNA
duplex called origins of replication
o Prokaryotes usually only have one origin of
replication whereas eukaryotes have multiple
origins
Unwinding causes a replication “bubble” at the
replication site
Origins tend to be AT rich because the weaker AT
bonds are easier to break because they only have 2 H bonds
The primer, which anneals to the template strand, is synthesized by a specific RNA polymerase
called primase. Primase involved RNA polymerization and does not require a primer
DNA polymerase will use this primer to extend the daughter strand
The unwinding of DNA by helicase can cause supercoiling ahead of the replication fork. These
supercoils are relieved by an enzyme called topoisomerase
Mutations of topoisomerase can be lethal
DNA replication occurs in the 5’ 3’ direction and the two strands of DNA are antiparallel.
One strand is able to synthesize a daughter strand in the direction of unwinding without
interruption. This strand is called the leading strand and undergoes continuous replication
The other strand, called the lagging strand, cannot synthesize a new strand in the 5’ 3’
direction and must be synthesized in interrupted fragments
Lagging strand synthesis must proceed by creating short fragments called Okazaki fragments
RNA primers anneal to the lagging strand
DNA polymerase elongates the strand until it hits the origin or another RNA primer
o Elongation occurs opposite to the direction of unwinding
The previous primer is removed and is replaced with dNTPs from DNA polymerase
o This leaves a break in the sugar phosphate backbone which must be repaired by
DNA ligase
The size of the Okazaki fragments depends on the species, or whether the organism is a
eukaryote or prokaryote
This process is true for both prokaryotes and eukaryotes
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