DNA REPLICATION
Cells are capable of giving rise to a new generation of cells by undergoing
DNA replication and cell division.
The process of DNA replication is composed of the following three basic
phases: the initiation phase, the elongation phase and the termination
phase.
Initiation phase
Replication begins when proteins bind at a specific site on the DNA known as
the replication origin. The closed circular DNA of prokaryotes usually has only
one such site, whereas the linear DNA of eukaryotes has multiple origins of
replication. Enzymes work together to expose the DNA, template strands.
DNA helicase unwinds the double helix by breaking the hydrogen bonds
between the complementary base pairs.
Because the base pairs are
complementary and have a natural
tendency to anneal, the two individual
strands are kept apart by singlestranded binding proteins (SSBs).
SSBs bind to the exposed DNA single
strands and block hydrogen bonding.
DNA gyrase relieves any tension brought
about by the unwinding of the DNA
strands during bacterial replication.
Replication begins in two directions from
the origin as a region of the DNA is
unwound. The DNA cannot be fully
unwound because of its large size
compared with the size of the cell. As the
two strands of DNA are disrupted, the
junction where they are still joined is called the replication fork. DNA
replication proceeds toward the direction of the replication fork on one
strand and away from the fork on the other strand. In eukaryotes, more than
one replication fork may exist on a DNA molecule at once because of the
multiple sites of origin, ensuring the rapid
replication of DNA. When two replication forks
are near each other, a replication bubble
forms.
MULTIPLE SITES ACCELERATE REPLICATION
In eukaryotes, DNA replication occurs at more than one site at a time, resulting in hundreds of
replication forks across a DNA strand. Eventually the replication bubbles become continuous
and the two new double-stranded daughter molecules are completely formed.
DNA polymerase III adds nucleotides at a rate of 50 per second in humans. At this rate, it
would take approximately 700 days to replicate the 3 billion base pairs of the human genome,
yet the whole process takes place in approximately 5 to 10 hours. The accelerated rate
indicates that replication is occurring at many points at once.
Elongation Phase
In prokaryotes, DNA
polymerase I, II, and III
function in replication and
repair. The enzyme that
builds the complementary
strand using the template
strand as a guide in
prokaryotes is DNA
polymerase III. DNA
polymerase III functions
only under certain
conditions. In order to
synthesize the new strand
in the 5’ to 3’ direction
DNA polymerase reads the template strand in the 3’ to 5’ or opposite
direction.
DNA polymerase elongates a polynucleotide strand, but it
cannot start a strand from scratch. Thus a short strand of ribonucleic acid
known as an RNA primer must be available to serve as a starting point for
the attachment of new nucleotides.
The primer is synthesized by the
enzyme primase.
The free bases in the nucleoplasm
used by DNA polymerase III to build
complementary strands are
deoxyribonucleotide triphosphates.
DNA polymerase III uses the energy
derived from breaking the bond
between the 1st and 2nd phosphate to
drive the dehydration synthesis that
adds a complementary nucleotide to
the elongating strand. The extra 2
phosphates are recycled by the cell.
Both newly synthesized DNA strands
are given different names. The
leading strand is built continuously
toward the replication fork. The
lagging strand is built discontinuously
away from the replication fork. The
discontinuous sections are called
Okazaki fragments. RNA primers are
continuously added as the lagging
strand is being built. DNA polymerase
I removes the RNA primers and replaces them with appropriate
deoxyribonucleotides by extending the preceding Okazaki fragment. DNA
ligase, joins the Okazaki fragments into one strand by creation of a
phosphodiester bond.
Termination Phase
The newly formed daughter DNA molecules rewind automatically without the
help of any enzyme activity.
DNA polymerase III and DNA polymerase I can also function as exonucleases
– these enzymes proofread the newly synthesized strand. When mistakes
occur, the enzymes cut out the incorrect nucleotides. For example, if a
thymine is added across from a cytosine, they cannot form hydrogen bonds
and the strand is unstable. DNA polymerase III can’t move forward if base
pairs are mismatched. DNA polymerase III will back up, replaces the
incorrect base with the correct one and continues on.
After a strand has been replicated, rare mismatching errors may still occurusually an average of 1 error for every million base pairs. However, even this
number of errors can have serious implications for an organism. Special DNA
repair mechanisms read the strands for errors that might have been missed by
DNA polymerase III. These repair mechanisms are complexes of proteins and
enzymes. Enzymes remove a portion of the strand around the mismatch. The
resultant gap is filled by in by a DNA polymerase and completed with DNA
ligase. Similar repair mechanisms help to correct damage caused by
chemicals and radiation, including ultraviolet light. Without these repair
mechanisms, DNA would accumulate many errors.
Nucleotide Excision Repair Animation
Telomeres
Each end of a linear chromosome presents
a problem for the DNA replication process.
Once the RNA primer has been removed
from the 5’ end of each daughter strand,
there is no adjacent fragment onto which
new DNA nucleotides can be added to fill
the gap. Therefore, each replication
results in a slightly shorter chromosome and
the loss of DNA at the end of
chromosomes. Furthermore, the
nucleotides on the complementary strand
are left unpaired, and they eventually
break off from the new strand. If this loss of
a DNA tip through lack of replication was
not prevented in some way, chromosomes
would continue to shorten during each
replication cycle. Special regions at the
end of each chromosome in eukaryotes
help to guard against this problem. These
regions, called telomeres, serve as a buffer zone. Telomeres are sequences
of repetitive DNA that do not direct cell development. The telomeres of
human chromosomes have 250 to 1500 repetitions of the base sequence
TTAGGG.
If cells divided without telomeres, they would lose the end of their
chromosomes, and the necessary information they contain. The erosion of
telomeres is related to cell death and aging. Conversely, the extension of
telomeres is linked to a longer life span for the cell.