DNA synthesis: molecular events that allow assembly of new DNA strands
DNA replication: the copying of double stranded DNA, necessary for cell
reproduction before cell division; involves DNA chain synthesis, initiation, elongation,
termination and daughter strand separation. DNA replication occurs in the ‘S’ phase
of the cell cycle
DNA replication is semi-conservative – proposed by Watson and Crick – the 2
parental strands separate and each makes a copy of itself. After 1 round of
replication, the 2 daughter molecules each comprises 1 old and 1 new strand. After 2
rounds, 2 out of the 4 new molecules contain 1 old and 1 new while 2 consist only of
new material.
Conservative and dispersive replication were still plausible until the theories could be
disproved…
- Conservative replication: after 1 round of replication, 1 DNA molecule will
consist of 2 new strands and 1 molecule will consist of 2 old/original stands
- Dispersive replication: the old material is split in chunks and distributed
between the 2 daughter molecules
In 1958, Meselson and Stahl devised an experiment to determine whether DNA
replication is conservative, semi-conservative, or dispersive. For the experiment,
1. E. coli were grown in a medium with the heavier N15 isotope
2. E. coli with only N15 in their DNA were transferred to a N14 medium and left
to divide
3. DNA was extracted at a specific time and solved in caesium chloride then
centrifuged on a salt density gradient to separate/isolate the DNA
,100% of the DNA is composed of 1 light strand and 1 heavy strand after 1
generation of growth in N-14 containing growth media.
After 40 minutes, the second cell doubling, there are 2 types of the DNA, a lighter
(no N15) and a half heavy (containing 1 strand of N15, as 2 different bands are
obtained.
Origin of replication (ORI): A unique DNA sequence of a replicon where DNA
replication is initiated and proceeds typically bidirectionally.
The point at which replication occurs is called the ‘replication fork’.
The replication fork: the site of the progressive unwinding of the double-stranded
DNA and the duplication of the template DNA, it travels along the length of the DNA
molecule, away from the ORI.
In a circular DNA molecule (bacterial DNA), there is a single ORI that spreads
bidirectionally throughout the molecule. Replication proceed until the eye occupy the
whole chromosome.
In a linear DNA molecule (a chromosome), there are multiple ORI that spread bi-
directionally along the chromosome, away from the ORI, until they reach another
replication fork moving in the opposite direction.
The ORI in prokaryotes is called the ‘OriC’ and it is a well defines structure. It is
245bp in length and contains…
- AT rich region
- DNAa boxes
- GATC methylation sites (methylation of the bacterial origin regulates initiation)
DNA sequences at the replication origins in Higher Eukaryotes, including humans
vary and have been much harder to isolate.
DNA replication starts with an RNA primer as DNA polymerase requires a RNA
primer to begin the polymerisation. DNA synthesis occurs 5’ to 3’. One strand is
, synthesised as a continuous molecule (leading strand), but the other is made in short
sections/pieces called ‘Okazaki fragments’ (lagging strand/discontinuous).
- The leading strand is the strand being synthesized in the same direction as
the growing replication fork. A polymerase "reads" the leading strand template
and adds complementary nucleotides to the nascent leading strand
continuously
- The lagging strand is synthesizes in opposite to the direction of the growing
replication fork. Because of its orientation, replication of the lagging strand is
more complicated than that of the leading strand. On the lagging strand
template, a primase "reads" the template DNA and initiates synthesis of a
short complementary RNA primer. A DNA polymerase extends the primed
segments, forming Okazaki fragments
Replication terminates when the two
growing forks meet in the terminus
region, which is located 180º around
the circular chromosome from the
ORI. Replication
Fork 2
The termination sequences in E. coli
are called ‘ter’ sites, which serve as Replication
binding sites for the ‘Tus terminator’ Fork 1
protein. Tus can block the progression
of replication forks in specific
directions. Replication is terminated in
bacterial systems by a "replication
fork trap", studded with termination
sites which causes the bidirectional
forks to pause, encounter and fuse
within a region called the ‘terminus
region’.
Forks meet here
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