REPLICATION OF DNA

• The process of DNA duplication is called DNA replication. Replication

follows several steps that involve multiple proteins called replication enzymes
and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA
replication occurs in the S phase of interphase during the cell cycle. The
process of DNA replication is vital for cell growth, repair, and reproduction
in organisms.
 DNA Structure
• DNA or deoxyribonucleic acid is a type of molecule known as a nucleic acid.
It consists of a 5-carbon deoxyribose sugar, a phosphate, and a nitrogenous
base. Double-stranded DNA consists of two spiral nucleic acid chains that
are twisted into a double helix shape. This twisting allows DNA to be more
compact. In order to fit within the nucleus, DNA is packed into tightly coiled
structures called chromatin. Chromatin condenses to form chromosomes
during cell division. Prior to DNA replication, the chromatin loosens giving
cell replication machinery access to the DNA strands.
 Preparation For Replication
Step 1: Replication Fork Formation
Before DNA can be replicated, the double stranded molecule must
be “unzipped” into two single strands. DNA has four bases called
adenine (A), thymine (T), cytosine (C) and guanine (G) that form
pairs between the two strands. Adenine only pairs with thymine and
cytosine only binds with guanine. In order to unwind DNA, these
interactions between base pairs must be broken. This is performed
by an enzyme known as DNA helicase. DNA helicase disrupts the
hydrogen bonding between base pairs to separate the strands into a
Y shape known as the replication fork. This area will be the
template for replication to begin.

DNA is directional in both strands, signified by a 5' and 3' end. This
notation signifies which side group is attached the DNA backbone.
The 5' end has a phosphate (P) group attached, while the 3' end has
a hydroxyl (OH) group attached. This directionality is important for
replication as it only progresses in the 5' to 3' direction. However,
the replication fork is bi-directional; one strand is oriented in the 3'
to 5' direction (leading strand) while the other is oriented 5' to 3'
(lagging strand). The two sides are therefore replicated with two
different processes to accommodate the directional difference.
Replication Begins
 Step 2: Primer Binding
The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece
of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point
for replication. Primers are generated by the enzyme DNA primase.
 DNA Replication: Elongation
Step 3: Elongation
Enzymes known as DNA polymerases are responsible creating the new
strand by a process called elongation. There are five different known
types of DNA polymerases in bacteria and human cells. In bacteria such
as E. coli, polymerase III is the main replication enzyme, while
polymerase I, II, IV and V are responsible for error checking and repair.
DNA polymerase III binds to the strand at the site of the primer and
begins adding new base pairs complementary to the strand during
replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are
the primary polymerases involved in DNA replication. Because
replication proceeds in the 5' to 3' direction on the leading strand, the
newly formed strand is continuous.

The lagging strand begins replication by binding with multiple primers.

Each primer is only several bases apart. DNA polymerase then adds
pieces of DNA, called Okazaki fragments, to the strand between primers.
This process of replication is discontinuous as the newly created
fragments are disjointed.
 Step 4: Termination

Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all
RNA primers from the original strands. These primers are then replaced with appropriate bases. Another
exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme
called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear
DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the
parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the
end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase
enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once
completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In
the end, replication produces two DNA molecules, each with one strand from the parent molecule and one
new strand.
Replication Enzymes
 DNA replication would not occur without enzymes that catalyze
various steps in the process. Enzymes that participate in the
eukaryotic DNA replication process include:
• DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It
forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
• DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA
molecules that act as templates for the starting point of DNA replication.
• DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and lagging
DNA strands.
• Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from
becoming tangled or supercoiled.
• Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain.
• DNA ligase - joins DNA fragments together by forming phosphodiester bonds between
nucleotides.
 DNA Replication Summary
• DNA replication is the production of identical DNA helices from a single double-
stranded DNA molecule. Each molecule consists of a strand from the original
molecule and a newly formed strand. Prior to replication, the DNA uncoils and
strands separate. A replication fork is formed which serves as a template for
replication. Primers bind to the DNA and DNA polymerases add new nucleotide
sequences in the 5′ to 3′ direction. This addition is continuous in the leading strand
and fragmented in the lagging strand. Once elongation of the DNA strands is
complete, the strands are checked for errors, repairs are made, and telomere
sequences are added to the ends of the DNA.
 RNA Structure
• RNA is typically single stranded and is made of ribonucleotides that are linked by
phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the
pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a
phosphate group. The subtle structural difference between the sugars gives DNA
added stability, making DNA more suitable for storage of genetic information,
whereas the relative instability of RNA makes it more suitable for its more short-
term functions. The RNA-specific pyrimidine uracil forms a complementary base
pair with adenine and is used instead of the thymine used in DNA. Even though
RNA is single stranded, most types of RNA molecules show extensive
intramolecular base pairing between complementary sequences within the RNA
strand, creating a predictable three-dimensional structure essential for their function
(Figure 1 and Figure 2).
 Functions of RNA in Protein Synthesis
• Cells access the information stored in DNA by creating RNA to direct the
synthesis of proteins through the process of translation. Proteins within a
cell have many functions, including building cellular structures and serving as
enzyme catalysts for cellular chemical reactions that give cells their specific
characteristics. The three main types of RNA directly involved in protein
synthesis are messenger RNA (mRNA), ribosomal RNA (rRNA), and
transfer RNA (tRNA).
• In 1961, French scientists François Jacob and Jacques Monod hypothesized
the existence of an intermediary between DNA and its protein products,
which they called messenger RNA.[1] Evidence supporting their hypothesis
was gathered soon afterwards showing that information from DNA is
transmitted to the ribosome for protein synthesis using mRNA. If DNA
serves as the complete library of cellular information, mRNA serves as a
photocopy of specific information needed at a particular point in time that
serves as the instructions to make a protein.
• The mRNA carries the message from the DNA, which controls all of the
cellular activities in a cell. If a cell requires a certain protein to be
synthesized, the gene for this product is “turned on” and the mRNA is
synthesized through the process of transcription (see RNA Transcription).
The mRNA then interacts with ribosomes and other cellular machinery
(Figure 3) to direct the synthesis of the protein it encodes during the process
of translation (see Protein Synthesis). mRNA is relatively unstable and short-
lived in the cell, especially in prokaryotic cells, ensuring that proteins are only
made when needed.
• rRNA and tRNA are stable types of RNA. In prokaryotes and eukaryotes,
tRNA and rRNA are encoded in the DNA, then copied into long RNA
molecules that are cut to release smaller fragments containing the individual
mature RNA species. In eukaryotes, synthesis, cutting, and assembly of
rRNA into ribosomes takes place in the nucleolus region of the nucleus, but
these activities occur in the cytoplasm of prokaryotes. Neither of these types
of RNA carries instructions to direct the synthesis of a polypeptide, but they
play other important roles in protein synthesis.
• Ribosomes are composed of rRNA and protein. As its name suggests, rRNA is a major
constituent of ribosomes, composing up to about 60% of the ribosome by mass and
providing the location where the mRNA binds. The rRNA ensures the proper alignment of
the mRNA, tRNA, and the ribosomes; the rRNA of the ribosome also has an enzymatic
activity (peptidyl transferase) and catalyzes the formation of the peptide bonds between two
aligned amino acids during protein synthesis. Although rRNA had long been thought to
serve primarily a structural role, its catalytic role within the ribosome was proven in 2000.[2]
Scientists in the laboratories of Thomas Steitz (1940–) and Peter Moore (1939–) at Yale
University were able to crystallize the ribosome structure from Haloarcula marismortui, a
halophilic archaeon isolated from the Dead Sea. Because of the importance of this work,
Steitz shared the 2009 Nobel Prize in Chemistry with other scientists who made significant
contributions to the understanding of ribosome structure.
 Transfer RNA is the third main type of RNA and one of the smallest, usually only 70–90 nucleotides
long. It carries the correct amino acid to the site of protein synthesis in the ribosome. It is the base pairing
between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide
chain being synthesized (Figure 4). Any mutations in the tRNA or rRNA can result in global problems for
the cell because both are necessary for proper protein synthesis (Table 1).
Structure and Function
of RNA mRNA rRNA tRNA

Short, unstable, single- Longer, stable RNA Short (70-90 nucleotides),

stranded RNA molecules composing stable RNA with extensive
Structure corresponding to a gene 60% of ribosome’s mass intramolecular base
encoded within DNA pairing; contains an amino
acid binding site and an
mRNA binding site
Serves as intermediary Ensures the proper Carries the correct amino
between DNA and alignment of mRNA, acid to the site of protein
Function protein; used by ribosome tRNA, and ribosome synthesis in the ribosome
to direct synthesis of during protein synthesis;
protein it encodes catalyzes peptide bond
formation between amino
acids