DNA Replication

One major question for the human mind is how life continues. One of
the most important mechanisms for all life cells to give offsprings is
undoubtedly the DNA Replication. DNA Replication answers to the
question: "When a cell divides, where the extra DNA comes from?"
What "DNA Replication" is? It is the process that can duplicate the DNA
of a cell.
Every cell (of eukaryotes or prokaryotes) has one or more DNA (or
RNA) polymer molecules that need to duplicate in order the cell
duplication to take place.
In the eukaryotes (organisms with cell that have nucleus) the DNA is
formed in two strands, each composed of units called Nucleotides. The
two strands look like two chains that form the DNA Double Helix.
The DNA Replication Process is capable of opening the Double Helix
and separating the two strands. Then the two strands are copied. As a
result two new DNA molecules are created. The next step is the cell
division. After that a daughter cell is created. In its nucleus lies a copy
of the parental DNA.
Chromosomes
A chromosome is an organized structure of DNA and protein that is
found in cells. It is a single piece of coiled DNA containing many genes,
regulatory elements and other nucleotide sequences. Chromosomes
also contain DNA-bound proteins, which serve to package the DNA and
control its functions. Chromosomes vary widely between different
organisms. The DNA molecule may be circular or linear, and can be
composed of 10,000 to 1,000,000,000 nucleotides in a long chain.
Typically eukaryotic cells (cells with nuclei) have large linear
chromosomes and prokaryotic cells (cells without defined nuclei) have
smaller circular chromosomes, although there are many exceptions to
this rule. Furthermore, cells may contain more than one type of
chromosome; for example, mitochondria in most eukaryotes and
chloroplasts in plants have their own small chromosomes.

Chromosomes in Eukaryotes

In eukaryotes, nuclear chromosomes are packaged by proteins into a

condensed structure called chromatin. This allows the very long DNA
molecules to fit into the cell nucleus. The structure of chromosomes
and chromatin varies through the cell cycle. Chromosomes are the
essential unit for cellular division and must be replicated, divided, and
passed successfully to their daughter cells so as to ensure the genetic
diversity and survival of their progeny. Chromosomes may exist as
either duplicated or unduplicated—unduplicated chromosomes are
single linear strands, whereas duplicated chromosomes (copied during
synthesis phase) contain two copies joined by a centromere.
Compaction of the duplicated chromosomes during mitosis and meiosis
results in the classic four-arm structure (pictured to the right).
Chromosomal recombination plays a vital role in genetic diversity. If
these structures are manipulated incorrectly, through processes known
as chromosomal instability and translocation, the cell may undergo
mitotic catastrophe and die, or it may aberrantly evade apoptosis
leading to the progression of cancer. Eukaryotes (cells with nuclei such
as those found in plants, yeast, and animals) possess multiple large
linear chromosomes contained in the cell's nucleus. Each chromosome
has one centromere, with one or two arms projecting from the
centromere, although, under most circumstances, these arms are not
visible as such. In addition, most eukaryotes have a small circular
mitochondrial genome, and some eukaryotes may have additional
small circular or linear cytoplasmic chromosomes.

In the nuclear chromosomes of eukaryotes, the uncondensed DNA

exists in a semi-ordered structure, where it is wrapped around
histones (structural proteins), forming a composite material called
chromatin.

Chromosomes in prokaryotes

The prokaryotes – bacteria and archaea – typically have a single

circular chromosome, but many variations do exist. Most bacteria have
a single circular chromosome that can range in size from only 160,000
base pairs in the endosymbiotic bacterium Candidatus Carsonella
ruddii to 12,200,000 base pairs in the soil-dwelling bacterium
Sorangium cellulosum Spirochaetes of the genus Borrelia are a notable
exception to this arrangement, with bacteria such as Borrelia
burgdorferi, the cause of Lyme disease, containing a single linear
chromosome.
DNA Replication

Replication of DNA occurs during the process of normal cell division

cycles. Because the genetic complement of the resultant daughter cells
must be the same as the parental cell, DNA replication must possess a
very high degree of fidelity. The entire process of DNA replication is
complex and involves multiple enzymatic activities.

DNA Polymerase

A DNA polymerase is an enzyme that catalyzes the polymerization of

deoxyribonucleotides into a DNA strand. DNA polymerases are best-
known for their role in DNA replication, in which the polymerase
"reads" an intact DNA strand as a template and uses it to synthesize
the new strand. This process copies a piece of DNA. The newly-
polymerized molecule is complementary to the template strand and
identical to the template's original partner strand. DNA polymerases
use a magnesium ion for catalytic activity.

Prokaryotic DNA polymerases

Bacteria have 3 known DNA polymerases:

 Pol I: implicated in DNA repair; has 5'-3' (Polymerase) activity

and both 3'-5' exonuclease (Proofreading) and 5'-3' exonuclease
activity (RNA Primer removal).
 Pol II: involved in reparation of damaged DNA; has 3'-5'
exonuclease activity.
 Pol III: the main polymerase in bacteria (elongates in DNA
replication); has 3'-5' exonuclease proofreading ability.

Eukaryotic DNA polymerases

Eukaryotes have at least 5 DNA Polymerases:

 Pol α (also called RNA primase): acting as a primase

(synthesizing an RNA primer), elongating that primer with DNA
nucleotides. After around 20 nucleotides elongation is taken over
by Pol δ.
 Pol β: Implicated in repairing DNA, in base excision repair and
gap-filling synthesis.
 Pol γ: Replicates and repairs mitochondrial DNA and has
proofreading 3'-5' exonuclease activity.
  Pol δ: Highly processive and has proofreading 3'-5' exonuclease
activity. The main polymerase involved in synthesis, though
there is still debate about its role.
 Pol ε: Also highly processive and has proofreading 3'-5'
exonuclease activity. Highly related to pol δ, and thought to be
the main polymerase involved in leading strand synthesis,
though there is again still debate about its role.

DNA Replication models

The process of DNA Replication was hiding many secrets. One of the
most important was how the two daughter strands are created. The
DNA is a complex of two chains. In order the hereditary phenomenon
to be explained, these strands should be accurately copied and
transmitted from the parental cell to the daughter ones. These are
three possible models that describe the accurate creation of the
daughter chains:

1) Semiconservative Replication According to this model, DNA

Replication would create two molecules. Each of them would be a
complex of an old (parental and a daughter strand).

2) Conservative Replication According to this model, the DNA

Replication process would create a brand new DNA double helix made
of two daughter strands while the parental chains would stay together.

3) Dispersive Replication According to this model the Replication

Process would create two DNA double-chains, each of them with parts
of both parent and daughter molecules.
Meselson–Stahl experiment

The Meselson–Stahl experiment was an experiment by Matthew

Meselson and Franklin Stahl in 1958 which supported the hypothesis
that DNA replication was semiconservative. Semiconservative
replication means that when the double stranded DNA helix was
replicated, each of the two double stranded DNA helices consisted of
one strand coming from the original helix and one newly synthesized.
It has been called "the most beautiful experiment in biology.

Three hypotheses had been previously proposed for the method of

replication of DNA.

In the semiconservative hypothesis, proposed by Watson and Crick,

the two strands of a DNA molecule separate during replication. Each
strand then acts as a template for synthesis of a new strand.

The conservative hypothesis proposed that the entire DNA molecule

acted as a template for synthesis of an entirely new one. According to
this model, histone proteins bound to the DNA, distorting it in such a
way as to expose both strands' bases for hydrogen bonding.

The dispersive hypothesis is exemplified by a model proposed by Max

Delbrück, which attempts to solve the problem of unwinding the two
strands of the double helix by a mechanism that breaks the DNA
backbone every 10 nucleotides or so, untwists the molecule, and
attaches the old strand to the end of the newly synthesized one. This
would synthesize the DNA in short pieces alternating from one strand
to the other.

Each of these three models makes a different prediction about the

distribution of the "old" DNA in molecules formed after replication. In
the conservative hypothesis, after replication, one molecule is the
entirely conserved "old" molecule, and the other is all newly
synthesized DNA. The semiconservative hypothesis predicts that each
molecule after replication will contain one old and one new strand. The
dispersive model predicts that each strand of each new molecule will
contain a mixture of old and new DNA.

Experiment

Nitrogen is a major constituent of DNA. 14N is by far the most

abundant isotope of nitrogen, but DNA with the heavier (but non-
radioactive) 15N isotope is also functional.
E. coli were grown for several generations in a medium with 15N. When
DNA is extracted from these cells and centrifuged on a salt density
gradient, the DNA separates out at the point at which its density
equals that of the salt solution. The DNA of the cells grown in 15N
medium had a higher density than cells grown in normal 14N medium.
After that, E. coli cells with only 15N in their DNA were transferred to a
14
N medium and were allowed to divide; the progress of cell division
was monitored by measuring the optical density of the cell suspension.

DNA was extracted periodically and was compared to pure 14N DNA
and 15N DNA. After one replication, the DNA was found to have close to
the intermediate density. Since conservative replication would result in
equal amounts of DNA of the higher and lower densities (but no DNA
of an intermediate density), conservative replication was excluded.
However, this result was consistent with both semiconservative and
dispersive replication. Semiconservative replication would result in
double-stranded DNA with one strand of 15N DNA, and one of 14N DNA,
while dispersive replication would result in double-stranded DNA with
both strands having mixtures of 15N and 14N DNA, either of which
would have appeared as DNA of an intermediate density.

The authors continued to sample cells as replication continued. DNA

from cells after two replications had been completed was found to
consist of equal amounts of DNA with two different densities, one
corresponding to the intermediate density of DNA of cells grown for
only one division in 14N medium, the other corresponding to DNA from
cells grown exclusively in 14N medium. This was inconsistent with
dispersive replication, which would have resulted in a single density,
lower than the intermediate density of the one-generation cells, but
still higher than cells grown only in 14N DNA medium, as the original
15
N DNA would have been split evenly among all DNA strands. The
result was consistent with the semiconservative replication hypothesis.

Process

The ability of DNA polymerases to replicate DNA requires a number of

additional accessory proteins. The combination of polymerases with
several of the accessory proteins yields an activity identified as DNA
polymerase holoenzyme. These accessory proteins include:
1. Primase

2. Single strand binding proteins

3. Helicase

4. DNA ligase

5. Topoisomerases

Initiation of replication

Prokaryotes

In prokaryotes there is single origin of replication known as Ori C. It

contains a highly conserved 9 bp consensus sequence 5' - TTATCCACA
- 3'. On these sequence the helicase protein binds to melt or unwind
the DNA. The Helicase enzyme in prokaryotes is known as Dna B
protein. As soon as it unwinds the Double stranded structure of the
DNA, the SSBs (Single Strand Binding Proteins) comes and binds
to the two strands created in the replication fork.
Eukaryotes
In eukaryotes there are no. of origins of replication because of the
large amount and size of the DNA. The origin of replication is termed
as ARS (Autonomous replicating sequence). It contains four
regions (A, B1, B2, and B3) which are responsible for the melting or
unwinding of the double helical structure of the DNA. In this ARS, ABF
(autonomous replicating sequence binding factor) binds at B3 and
melting occurs at B2. A and B1 acts as the guiding portions.

Elongation

The process of DNA replication begins at specific sites in the

chromosomes termed origins of replication, requires a primer bearing
a free 3'–OH, proceeds specifically in the 5'—3' direction on both
strands of DNA concurrently and results in the copying of the template
strands in a semi conservative manner. The semi conservative
nature of DNA replication means that the newly synthesized daughter
strands remain associated with their respective parental template
strands.

The large size of eukaryotic chromosomes and the limits of nucleotide

incorporation during DNA synthesis, make it necessary for multiple
origins of replication to exist in order to complete replication in a
reasonable period of time. The precise nature of origins of replication
in higher eukaryotic organisms is unclear. However, it is clear that at a
replication origin the strands of DNA must dissociate and unwind in
order to allow access to DNA polymerase. Unwinding of the duplex at
the origin as well as along the strands as the replication process
proceeds is carried out by helicases. The resultant regions of single-
stranded DNA are stabilized by the binding of single-strand binding
proteins. The stabilized single-stranded regions are then accessible to
the enzymatic activities required for replication to proceed. The site of
the unwound template strands is termed the replication fork.
In order for DNA polymerases to synthesize DNA they must encounter
a free 3'–OH which is the substrate for attachment of the 5'–phosphate
of the incoming nucleotide. During repair of damaged DNA the 3'–OH
can arise from the hydrolysis of the backbone of one of the two
strands. During replication the 3'–OH is supplied through the use of an
RNA primer, synthesized by the primase activity. The primase utilizes
the DNA strands as templates and synthesizes a short stretch of RNA
generating a primer for DNA polymerase.

Synthesis of DNA proceeds in the 5'—3' direction through the

attachment of the 5'–phosphate of an incoming dNTP to the existing
3'–OH in the elongating DNA strands with the concomitant release of
pyrophosphate. Initiation of synthesis, at origins of replication, occurs
simultaneously on both strands of DNA. Synthesis then proceeds
bidirectionally, with one strand in each direction being copied
continuously and one strand in each direction being copied
discontinuously. During the process of DNA polymerases incorporating
dNTPs into DNA in the 5'—3' direction they are moving in the 3'—5'
direction with respect to the template strand. In order for DNA
synthesis to occur simultaneously on both template strands as well as
bidirectionally one strand appears to be synthesized in the 3'—5'
direction. In actuality one strand of newly synthesized DNA is
produced discontinuously.

The strand of DNA synthesized continuously is termed the leading

strand and the discontinuous strand is termed the lagging strand. The
lagging strand of DNA is composed of short stretches of RNA primer
plus newly synthesized DNA approximately 100–200 bases long (the
approximate distance between adjacent nucleosomes). The lagging
strands of DNA are also called Okazaki fragments. The concept of
continuous strand synthesis is somewhat of a misnomer since DNA
polymerases do not remain associated with a template strand
indefinitely. The ability of a particular polymerase to remain associated
with the template strand is termed its' processivity. The longer it
associates the higher the processivity of the enzyme. DNA polymerase
processivity is enhanced by additional protein activities of the
replisome identified as processivity accessory proteins.
Termination of replication

Because bacteria have circular chromosomes, termination of

replication occurs when the two replication forks meet each other on
the opposite end of the parental chromosome. E coli regulate this
process through the use of termination sequences which, when bound
by the Tus protein, enable only one direction of replication fork to pass
through. As a result, the replication forks are constrained to always
meet within the termination region of the chromosome.
Eukaryotes initiate DNA replication at multiple points in the
chromosome, so replication forks meet and terminate at many points
in the chromosome; these are not known to be regulated in any
particular manner. Because eukaryotes have linear chromosomes, DNA
replication often fails to synthesize to the very end of the
chromosomes (telomeres), resulting in telomere shortening. This is a
normal process in somatic cells — cells are only able to divide a certain
number of times before the DNA loss prevents further division. (This is
known as the Hayflick limit.) Within the germ cell line, which passes
DNA to the next generation, telomerase extends the repetitive
sequences of the telomere region to prevent degradation. Telomerase
can become mistakenly active in somatic cells, sometimes leading to
cancer formation.

Telomere formation and Telomerase enzyme

It is not possible for DNA polymerase to replicate the end of a linear

DNA molecule completely in the ordinary way, and this has led to the
evolution of special DNA sequences, called telomeres, at the ends of
eucaryotic chromosomes. These sequences, which are similar in
organisms as diverse as protozoa, fungi, plants, and mammals, consist
of many tandem repeats of a short sequence that contains a block of
neighboring G nucleotides. I
The problem of replicating the ends of chromosomes is solved in an
ingenious way by an enzyme called telomerase. This enzyme
recognizes the G-rich strand of an existing telomere repeat sequence
and elongates it in the 5'-to-3' direction. In the absence of a
complementary DNA strand, the telomerase synthesizes a new copy of
the repeat using an RNA template that is a component of the enzyme
itself. The enzyme thus contains the information used to maintain the
characteristic telomere sequences. After several rounds of extension
by telomerase, replication of the chromosome end can be completed
using these extensions as a template for synthesis of the
complementary strand by DNA polymerase. Because the processes
that shorten and restore the telomere sequence are only
approximately balanced, each chromosome end contains a variable
number of the tandem repeats, which generally extend for hundreds of
nucleotide pairs.