DNA Replication

GROUP 3
What is DNA Replication?
• In molecular biology, DNA replication is the biological process of
producing two identical replicas of DNA from one
original DNA molecule. This process occurs in all living organisms and
is the basis for biological inheritance. The cell possesses the
distinctive property of division, which makes replication of DNA
essential
• DNA is made up of a double helix of two complementary strands. During
replication, these strands are separated. Each strand of the original DNA
molecule then serves as a template for the production of its counterpart, a
process referred to as semiconservative replication. As a result of semi-
conservative replication, the new helix will be composed of an original DNA
strand as well as a newly synthesized strand. Cellular proofreading and error-
checking mechanisms ensure near perfect fidelity for DNA replication
• In a cell, DNA replication begins at specific locations, or origins of replication, in
the genome.[4] Unwinding of DNA at the origin and synthesis of new strands,
accommodated by an enzyme known as helicase, results in replication
forks growing bi-directionally from the origin. A number of proteins are
associated with the replication fork to help in
the initiation and continuation of DNA synthesis. Most prominently, DNA
polymerase synthesizes the new strands by adding nucleotides that
complement each (template) strand. DNA replication occurs during the S-stage
of interphase
• DNA replication (DNA amplification) can also be performed in
vitro (artificially, outside a cell). DNA polymerases isolated from cells
and artificial DNA primers can be used to initiate DNA synthesis at
known sequences in a template DNA molecule. Polymerase chain
reaction (PCR), ligase chain reaction (LCR), and transcription-
mediated amplification (TMA) are examples
 DNA replication, like all
biological polymerization
processes, proceeds in three
enzymatically catalyzed and
coordinated steps: initiation,
elongation and termination
Initiation
• For a cell to divide, it must first replicate its DNA. This process is
initiated at particular points in the DNA, known as "origins", which are
targeted by initiator proteins. In E. coli this protein is DnaA; in yeast,
this is the origin recognition complex. Sequences used by initiator
proteins tend to be "AT-rich" (rich in adenine and thymine bases),
because A-T base pairs have two hydrogen bonds (rather than the three
formed in a C-G pair) and thus are easier to strand-separate. Once the
origin has been located, these initiators recruit other proteins and form
the pre-replication complex, which unwinds the double-stranded DNA
has been located, these initiators recruit other proteins and form
the pre-replication complex, which unwinds the double-stranded DNA.
Role of initiators for initiation of DNA replication.
Formation of pre-replication complex.
Elongation
•DNA polymerase has 5′–3′ activity. All known DNA replication systems require a free
3' hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3′ to 5′
direction whereas a new strand is synthesized in the 5′ to 3′ direction—this is often confused).
Four distinct mechanisms for DNA synthesis are recognized:
1.All cellular life forms and many DNA viruses, phages and plasmids use a primase to synthesize a
short RNA primer with a free 3′ OH group which is subsequently elongated by a DNA polymerase
2.The retroelements (including retroviruses) employ a transfer RNA that primes DNA replication by
providing a free 3′ OH that is used for elongation by the reverse transcriptase
3.In the adenoviruses and the φ29 family of bacteriophages, the 3' OH group is provided by the
side chain of an amino acid of the genome attached protein (the terminal protein) to which
nucleotides are added by the DNA polymerase to form a new strand
4.In the single stranded DNA viruses—a group that includes the circoviruses, the geminiviruses,
the parvoviruses and others—and also the many phages and plasmids that use the rolling circle
replication (RCR) mechanism, the RCR endonuclease creates a nick in the genome strand (single
stranded viruses) or one of the DNA strands (plasmids). The 5′ end of the nicked strand is
transferred to a tyrosine residue on the nuclease and the free 3′ OH group is then used by the
DNA polymerase to synthesize the new strand
Termination
• Eukaryotes initiate DNA replication at multiple points in the chromosome, so
replication forks meet and terminate at many points in the chromosome. Because
eukaryotes have linear chromosomes, DNA replication is unable to reach the very end
of the chromosomes. Due to this problem, DNA is lost each replication cycle from the
end of the chromosome. Telomeres are regions of repetitive DNA close to the ends
and help prevent loss of genes due to this shortening. Shortening of the telomeres is
a normal process in somatic cells. This shortens the telomeres of the daughter DNA
chromosome. As a result, cells can only 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.
Increased telomerase activity is one of the hallmarks of cancer
Replication fork
• The replication fork is a structure that forms within the nucleus during
DNA replication. It is created by helicases, which break the hydrogen
bonds holding the two DNA strands together. The resulting structure
has two branching "prongs", each one made up of a single strand of
DNA. These two strands serve as the template for the leading and
lagging strands, which will be created as DNA polymerase matches
complementary nucleotides to the templates; the templates may be
properly referred to as the leading strand template and the lagging
strand template.
Scheme of the replication fork.
a: template, b: leading strand, c: lagging strand, d:
replication fork, e: primer, f: Okazaki fragments
Many enzymes are involved in the DNA replication fork.
• Leading strand
• The leading strand is the strand of nascent DNA which is being synthesized in the
same direction as the growing replication fork. This sort of DNA replication is
continuous.
• Lagging strand
• The lagging strand is the strand of nascent DNA whose direction of synthesis is
opposite to the direction of the growing replication fork. Because of its
orientation, replication of the lagging strand is more complicated as compared to
that of the leading strand. As a consequence, the DNA polymerase on this strand
is seen to "lag behind" the other strand.
• The lagging strand is synthesized in short, separated segments. 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. The RNA primers are then removed and
replaced with DNA, and the fragments of DNA are joined together by DNA ligase.
DNA Replication Protiens
• At the replication fork, many replication enzymes assemble
on the DNA into a complex molecular machine called the
replisome. The following is a list of major DNA replication
enzymes that participate in the replisome
 Enzyme Function in DNA replication

Also known as helix destabilizing enzyme. Helicase separates the two strands of DNA
DNA Helicase at the Replication Fork behind the topoisomerase.

The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA in
the 5' to 3' direction during DNA replication. Also performs proof-reading and error
DNA Polymerase correction. There exist many different types of DNA Polymerase, each of which
perform different functions in different types of cells.

A protein which prevents elongating DNA polymerases from dissociating from the DNA
DNA clamp parent strand.

Bind to ssDNA and prevent the DNA double helix from re-annealing after DNA helicase
Single-Strand Binding (SSB) Proteins unwinds it, thus maintaining the strand separation, and facilitating the synthesis of the
nascent strand.

Topoisomerase Relaxes the DNA from its super-coiled nature.

DNA Gyrase Relieves strain of unwinding by DNA helicase; this is a specific type of topoisomerase
Re-anneals the semi-conservative strands and joins Okazaki Fragments of the lagging
DNA Ligase strand.

Provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesis of the
Primase new DNA strand.

Lengthens telomeric DNA by adding repetitive nucleotide sequences to the ends

Telomerase of eukaryotic chromosomes. This allows germ cells and stem cells to avoid the Hayflick
limit on cell division
Bacteria
• Most bacteria do not go through a well-defined cell cycle but instead
continuously copy their DNA; during rapid growth, this can result in
the concurrent occurrence of multiple rounds of replication.[30] In E.
coli, the best-characterized bacteria, DNA replication is regulated
through several mechanisms, including: the hemimethylation and
sequestering of the origin sequence, the ratio of adenosine
triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of
protein DnaA. All these control the binding of initiator proteins to the
origin sequences
Dam methylates adenine of GATC sites after replication
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