Molecular basis of inheritance lecture 2

Replication
While proposing the double helical structure for DNA, Watson and Crick had immediately
proposed a scheme for replication of DNA. To quote their original statement that is as follows:
It has not escaped our notice that the specific pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material (Watson and Crick, 1953).
The scheme suggested that the two strands would separate and act as a template for the synthesis
of new complementary strands. After the completion of replication, each DNA molecule would
have one parental and one newly synthesised strand. This scheme was termed a semiconservative
DNA replication.

Fig 1: Watson-Crick model for semiconservative DNA replication

The Machinery and the Enzymes
In living cells the process of replication requires a set of catalysts (enzymes). The main
enzyme is referred to as DNA-dependent DNA polymerase, since it uses a DNA template to
catalyse the polymerisation of deoxynucleotides. These enzymes are highly efficient enzymes as
they have to catalyse polymerisation of a large number of nucleotides in a very short time. E. coli

that has only 4.6 106 bp completes the process of replication within 38 minutes; that means the
average rate of polymerisation has to be approximately 2000 bp per second. Not only do these
polymerases have to be fast, but they also have to catalyse the reaction with high degree of
accuracy. Any mistake during replication would result into mutations. Furthermore, energetically
replication is a very expensive process. Deoxyribonucleoside triphosphates serve dual purposes.
In addition to acting as substrates, they provide energy for polymerization reaction (the two
terminal phosphates in a deoxynucleoside triphosphates are high-energy phosphates, same as in
case of ATP).
In addition to DNA-dependent DNA polymerases, many additional enzymes are required
to complete the process of replication with high degree of accuracy. For long DNA molecules,
since the two strands of DNA cannot be separated in its entire length (due to very high energy
requirement), the replication occur within a small opening of the DNA helix, referred to as
replication fork. The DNA-dependent DNA polymerases catalyse polymerisation only in one
direction, that is 5'-3'. This creates some additional complications at the replicating fork.
Consequently, on one strand (the template with polarity 3'-5'), the replication is continuous, while
on the other (the template with polarity 5'-3' ), it is discontinuous. The discontinuously synthesized
fragments are later joined by the enzyme DNA ligase.
The DNA polymerases on their own cannot initiate the process of replication. Also the
replication does not initiate randomly at any place in DNA. There is a definite region in E. coli
DNA where the replication originates. Such regions are termed as origin of replication.

Fig2: Replication fork.

DNA Replication is Semiconservative

When the replication process is complete, two DNA molecules identical to each other and
identical to the original have been produced. Each strand of the original molecule has remained
intact as it served as the template for the synthesis of a complementary strand.
This mode of replication is described as semi-conservative: one-half of each new molecule of DNA
is old; one-half new. Watson and Crick had suggested that this was the way the DNA would turn
out to be replicated. Proof of the model came from the experiments of Meselson and Stahl.
They grew E. coli is a medium using ammonium ions (NH4+) as the source of nitrogen for DNA
(as well as protein) synthesis.

14

N is the common isotope of nitrogen, but they could also use

ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N. After growing E. coli
for several generations in a medium containing 15NH4+, they found that the DNA of the cells was
heavier than normal because of the 15N atoms in it. The difference could be detected by extracting
DNA from the E. coli cells and spinning it in an ultracentrifuge. The density of the DNA
determines where it accumulates in the tube. Then they transferred more living cells that had been
growing in 15NH4+ to a medium containing ordinary ammonium ions (14NH4+) and allowed
them to divide just once.
The DNA in this new generation of cells was exactly intermediate in density between that of the
previous generation and the normal. This, in itself, is not surprising. It tells us no more than that
half the nitrogen atoms in the new DNA are

14

N and half are

15

N. It tells us nothing about their

arrangement in the molecules. However, when the bacteria were allowed to divide again in normal
ammonium ions (14NH4+), two distinct densities of DNA were formed: half the DNA was normal
and half was intermediate.

Fig 3: Experiments of Meselson and Stahl.

Replication, in this process a cell copies its DNA prior to dividing. The DNA is copied 5-3 by

DNA polymerases using single stranded DNA as a template. Replication is semiconservative. In

E coli DNA polymerases I and III proof read sequences ensuring a very low error rate.
DNA synthesis occurs at the replication fork. A helicase separates the double helix and single
strand binding protein keeps the strands separate. DNA is synthesized continuously on the leading
strand and discontinuously as segments on the lagging strand. In prokaryotes DNA synthesis by
DNA polymerase is initiated using the RNA primer. Primer sequence is replaced with DNA later
by DNA polymerase. In eukaryotes, DNA polymerase initiates DNA synthesis by its integral
primase activity. The leading and lagging strands are synthesized respectively by DNA
polymerases. DNA ligase joins the Okazaki fragments by a phosphodiester bond.
Circular bacterial DNA molecules and linear eukaryotic chromosomes are replicated differently.
Circular DNA molecules are replicated from a single origin. Replication forks progress in both
directions eventually meeting and merging. Unwinding of the double helix produces supercoiling

of circular DNA molecules which is removed by topoisomerase. Replication of circular DNA

produces interlocked daughter molecules which are separated by topoisomerase.
Eukaryotic chromosomes are replicated from multiple origins. Replication bubbles form and these
eventually meet and merge. Transcriptionally active regions are replicated first. Replication
requires DNA to be unwound from nucleosomes. Special strategies are required to replicate the
ends of chromosomes. Telomerase adds noncoding sequences that allow replication of
chromosome ends.
Replication
Replication is necessary so that the genetic information present in cells can be passed on to
daughter cells following cell division. The DNA is copied by enzymes called DNA polymerases.
These act on single stranded DNA synthesizing a new strand complementary to the original strand.
DNA synthesis always occurs in the 5-3; direction. Replication is said to be semi conservative.
This means that each copied DNA molecule contains one strand derived from the parent molecule
and one newly synthesized daughter strand.
The mechanism of DNA replication is very similar inmost organisms. Differences exist only with
respect to the enzymes and proteins involved. In prokaryotes two enzymes (DNA polymerase I
and II) are responsible for DNA synthesis where as in eukaryotes five enzymes are involved.
Replication needs to be very accurate because even a small error rate would result in the loss of
important genetic information after just a few cell divisions. Accuracy is ensured by the ability of
the DNA polymerases to check that the correct bases have been inserted in the newly synthesized
strand. This is achieved through the enzyme which allows them to remove incorrectly inserted
bases from newly synthesized DNA and replace them with the correct base. This is referred to as
proofreading ability. It is estimated that just one base in five billion is inserted incorrectly.
Replication fork
During replication the double helix of a cells entire DNA is progressively unwound producing
segments of single stranded DNA which can be copied by DNA polymerases. Unwinding of the
double helix begins at a distinct position called the replication origin and gradually progresses
along the molecule, usually in both directions. Replication origins usually contain sequences rich

in weak A-T base pairs. The region where the helix unwinds and new DNA is synthesized is called
the replication fork.
Separation of double helix This is achieved by the action of a helicase enzyme. Following
separation of the strands, single stranded binding protein attaches to the DNA and prevents the
double helix from reforming.
Synthesis of leading and lagging strand- synthesis of DNA by DNA polymerases occurs only in
the 5-3 direction. As the two strands of the double helix run in opposite directions. Slightly
different mechanisms are required to replicate each. One strand called the leading strand is copied
in the same direction as the unwinding helix and so can be synthesized continuously. The other
strand known as the lagging strand is synthesized in the opposite direction and must be copied
discontinuously. The lagging strand is synthesized as a series of segments known as Okazaki
fragments.
DNA polymerases require a short double stranded region to initiate or prime DNA synthesis. This
is produced by an RNA polymerase called primase, which is able to initiate synthesis on single
stranded DNA. The primase synthesize a short RNA primer sequence on the DNA template
creating a short double stranded region. In prokaryotes DNA polymerase then synthesize DNA
beginning at the RNA primer. On the lagging strand, synthesis ends when the next RNA primer
encountered. At this point DNA polymerase I takes over and removes the RNA primer replacing
it with DNA.
Ligation the final step required to complete synthesis of the lagging strand is for the Okazaki
fragments to be joined together by phosphodiester bonds. This is carried out by a DNA ligase
enzyme.
Prokaryotic replication
Although the mechanism of DNA is similar in all organisms, the overall process varies depending
on the nature of the DNA molecule being copied. Different strategies are required for replication
of the circular DNA molecules which occur in bacteria and for the linear chromosomal DNA
molecules present in eukaryotes. The simplest and most common form of replication for circular
DNA involves a single origin of replication from which two replication forks progress in opposite
directions. The replicating forks eventually meet and fuse and replication terminates.

The replication of DNA molecules requires unwinding of the DNA double helix. This causes the
helix ahead of the replication fork to rotate. For circular DNA molecules that do not have free
ends, this produces supercoiling of the DNA preventing the replication fork from progressing. This
problem is overcome by the action of enzyme called topoisomerase. DNA topoisomerase produces
a break in the polynucleotide backbone of one of the DNA strands a short distance ahead of the
replication fork, allowing the DNA to rotate freely around the other intact strand removing the
supercoiling. The enzyme then rejoins the ends of the broken strand. When replication of a
bacterial chromosome is completed, two circular daughter molecules produced.
Eukaryotic replication
Due to the extreme length of eukaryotic chromosomes, DNA replication must be initiated at
multiple origins to ensure that the process is completed within a reasonable time span. Replication
forks proceed in either direction from each replication origin forming replication bubbles. DNA
replicated from a single origin is called a replicon. A typical mammalian cell has 50- 100000

replicons, each of which replicates 40-200kb of DNA. Not all the DNA is replicated at once.
Clusters of about 50 replicons initiate simultaneously at defined points.
The DNA eukaryotic chromosomes is packaged as DNA- protein complexes. As the replication
fork progresses DNA must unwind from the complex fir replication to occur. This slows the
progress of the replication forks and may explain the short length of the Okazaki fragments on the
lagging strand in eukaryotes (100-200bases) compared with prokaryotes (1000-2000 bases).
Replication of linear eukaryotic chromosomes poses a problem not encountered with circular
bacterial chromosomes in that the extreme 5 end of the lagging strand cannot be replicated
because there is no room for an RNA primer to initiate replication. This creates the potential for
chromosomes to shorten after each round of replication leading to a loss of genetic information.
The problem is overcome by a specialized structured at the end of the chromosome known as the
telomere. The enzyme telomerase contains an RNA molecule which partly overlaps with and binds
to the repeat sequence on the leading strand. The enzyme then extends the leading strand using
RNA as a template. This process of extension may occur hundreds of times before the telomerase
finally dissociates.

A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary
transcripts. "Begin" indicates the 3' end of the DNA template strand, where new RNA synthesis
begins; "end" indicates the 5' end, where the primary transcripts are almost complete.

Some viruses (such as HIV), have the ability to transcribe RNA into DNA. HIV has an RNA
genome that is duplicated into DNA. The resulting DNA can be merged with the DNA genome of
the host cell.

Fig : Replication of DNA at telomeres

The main enzyme responsible for synthesis of DNA from an RNA template is called reverse
transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a
complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme,
ribonuclease H, digests the RNA strand, and reverse transcriptase synthesizes a complementary

strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's
genome via another enzyme (integrase) causing the host cell to generate viral proteins, which
reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death
(apoptosis).
Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase.
Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase
carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA. This
repeated sequence of "junk" DNA is important because every time a linear chromosome is
duplicated, it is shortened in length. With "junk" DNA at the ends of chromosomes, the shortening
eliminates some repeated, or junk sequence, rather than the protein-encoding DNA sequence that
is further away from the chromosome ends. Telomerase is often activated in cancer cells to enable
cancer cells to duplicate their genomes without losing important protein-coding DNA sequence.
Activation of telomerase could be part of the process that allows cancer cells to become technically
immortal.

Fig: Reverse transcriptase activity

Retroviruses
These are an important group of RNA viruses with single stranded positive sense RNA
genomes. The group includes HIV which causes acquired immune deficiency syndrome (AIDS).

Retroviruses contain two copies of the genome in each viral particle. On infection of a
host cell, the ssRNA enters the cell is converted to dsDNA copy by reverse transcriptase and is
integrated into the host cell genome by a viral integrase enzyme.