Process of Dna Replication (dna Replication Animation)

DNA replication is the process that occurs in all living organisms by which new DNA molecules
are synthesized. DNA Replication in living cells is through semiconservative process in which
the already existing DNA molecule acts as a template for the synthesis of new DNA molecule.
Steps in DNA replication (dna replication animation)
The first step in the process of DNA replication is unwinding of the double stranded DNA
molecule. The unwinding is catalyzed by the specific enzymes called helicases. During
unwinding process the hydrogen bonds between the strands are broken
Each single strand of DNA now serves as a template for the synthesis of new complimentary
DNA strand. DNA polymerases III enzyme perform the synthesis of new complimentary strand
as it moves along the template strand from 3 to 5 direction. DNA polymerase delta is enzyme
involved in the synthesis of new complimentary strand in eukaryotes. The base pair always
occurs between adenine and thymine and guanine and cytosine.
The leading strand is the DNA template strand of the double helix so that replication fork moves
in the 3' to 5' direction. The lagging strand is the strand of DNA template double helix that is
oriented so that replication fork moves in a 5' to 3' manner. Finally DNA ligase I enzyme stitches
the together the lagging strand.

ntroduction to summary of DNA replication:

Replication is the formation of exact replica or carbon copy. Copying of DNA to make more
DNA copies is called DNA replication. It is an autocatalytic function of DNA. Replication can
occur by three methods: conservative, disruptive and semi-conservative.
In each replica, one-half is parent structure and one-half new structure. During replication, the
two strands of DNA separate, function as templates and help in synthesis of their complementary
strands so that in each daughter DNA duplex one strand is old and one new.

Mechanism of Dna Replication:

Prokaryotic DNA acts as a single replicating unit called replicon. Euacryotic DNA has a number
of replicons or replicating units. Each replicon or replication unit has particular region where
replication starts. It is called origin of replication or ori. in region of ori, there is a particular
nucleotide sequence called automatic replicating sequence or ars.replication proceeds
bidirectionally from each ori unidirectional replication seems to be rare though cairns(1953)has
claimed it to occur in prokaryotes. A replication fork is produced on each side of ori. Replication
will continue till a replication fork meets another replication fork .the area often called
termination point. It makes DNA replication semidiscontinuous.

Activation of deoxyribonucleotides: in the presence of energy and enzyme

phosphorylase, dAMP, dGMP, dCMP, dTMP are changed into active triphosphate forms
dATP, dGTP, dCTP and dTTP.
Exposure of DNA strands: the two strands separates at the sites of origin of replication or
ori by means of enzyme helicase. The separated strands are stabilized by single strand
binding proteins. Unwinding produces a coiling tension which is reduce by enzymes
called topoisomerases. Unzipping or separation of two strands of DNA produces a Yshaped configuration known as replication fork. The whole DNA of a replicon does not
open in one stretch but separation proceeds slowly. The two separated strands now
function as templates. Initiation of replication occurs at their 3 ends.

DNA polymerase: common DNA synthesizing enzyme is DNA polymerase III. Others are
DNA polymerase I and DNA polymerase II in prokaryotes. In eukaryotes, there are five
types of DNA polymerases. All of them function in 5 to 3 direction. However, they also
possess exonuclease activity in 3 to 5 direction.

RNA primer: a small strand of RNA called primer is synthesized at the 3 end of each
template with the help of RNA polymerase enzyme called primase. The primer lies at the
free end of one template strand and fork end of the other template strand.

Base pairing: deoxyribonucleosides triphosphates come to lie opposite nitrogen bases of

the template strands with dATP opposite T, dTTP opposite A, dCTP opposite G and dGTP
opposite C. pyrophosphatase removes two phosphate radicals. Energy is released which
helps in establishing hydrogen bonds between the complementary nitrogen bases.
Strand formation: DNA polymerase III establishes phospodiester linkages between the
adjacent deoxyribonucleosides phosphates in the presence of ATP/GTP, TPP and Mg2+. It
produces a new DNA strand. RNA primer dissociates and deoxyribonucleotides fill up
the gap. DNA polymerase III binds nucleotides in 5to 3 direction. Replication process in
opposite direction on the two templates. As replication proceeds new regions of DNA
duplex unwind and separate. Because of sequential opening of parent DNA duplex and its
replication to form two DNA double chains, DNA replication is also called zipper
duplication. One new strand is formed continuously in 5 to 3direction as its template
opens in 3 to 5direction called leading (continuous) strand. The template for second new
strand opens in 5 to 3direction. The second new strand is formed in short segments or
Okazaki segments (3000 4000 nucleotides) because DNA polymerization can occur in
only 5 to 3direction which is from fork end to free end (reverse of the other strand). A
new RNA primer is required every time on Okazaki segment is to be formed. Okazaki
segments are joined by means of DNA ligase (Khorana, 1967). The strand formed by
joining Okazaki segments is called lagging (discontinuous) strand. RNA primer is
removed by DNA polymerase I, which also helps in the formation of
oligodeoxyribonucleotide chain in its place for completing the replica chain. Since one
chain formation is continuous while second chain formation is discontinuous, DNA
replication is called semidiscontinuous.

Conclusion of Summary of Dna Replication:

There s a system for removing mismatched nitrogen bases and introduction of appropriate bases.
DNA polymerase III performs the function of removing and sealing. A wrong segment can be
corrected by nicking with endonuclease, synthesis of a new correct segment by DNA polymerase
I (Kornberg, 1969) and sealing by DNA ligase.

Dna Replication in Eukaryotes

Eukaryotic DNA replication proceeds in same way as that of prokaryotic DNA replication except
for following aspects:
1. Eukaryotic DNA has multiple origin of replication sites, so several replication forks create
many bubbles along DNA length. These replication forks are formed at autonomously
replicating sequences (ARS) that contain degenerate 11-bp sequences known as origin
replication element (ORE). ORE is located adjacent to an 80-bp AT rich sequence that is easy to
unwind.
2. DNA polymerases and are main DNA replication enzymes in eukaryotic cell. DNA
polymerase has 5---> 3 polymerase activity and synthesize primer on lagging strand which
are then extended by multisubunit DNA polymerase. DNA polymerase has a 3 5
proofreading exonuclease activity and carry out both leading and lagging strand synthesis in a
complex comparable to dimeric bacterial DNA polymerase III.
DNA polymerase removes primers of Okazaki fragments on lagging strand. DNA polymerase
is responsible for replication of mt DNA.
3. Telomeres, structures at ends of linear eukaryotic chromosomes, consist of many tandem
copies of a short oligonucleotide sequence with TxGy in one strand and CyAx in complementary
strand, where x and y are typically in range of 1 to 4. Telomerase contains RNA that act as a
template for synthesis of TxGy strand of telomere.
Protein component of telomerase act as a cellular reverse transcriptase for RNA-dependent DNA
synthesis. After extension of TxGy strand by telomerase, complementary CyAx strand is
synthesized by cellular DNA polymerases, starting with an RNA primer.

Introduction
DNA contains all the genetic information of the cell. It sends all the genetic information through
a transcript called RNA, which comes out of the nucleus to order ribosome to start producing
different kinds of polypeptide chains. The DNA codons are converted to RNA codons during
transcription. Transcription is the process in which DNA is converted into a complementary
RNA, catalyzed by an enzyme called RNA polymerase. Transcription is very important because
the genetic material DNA cannot come out of the nucleus through nuclear pores to instruct the
ribosome.

Steps Involved in Transcription

Unwinding of DNA
RNA polymerase recognizes the promoter sequence and binds to it. The promoter region
identifies the start of a gene, which strand is to be copied, and the direction of copying of
strand.

Complementary bases are put against the bases on the template (But U is placed instead
of T).

Transcription will stop once it reaches the termination code on DNA.

The mRNA produced is called an mRNA transcript.

Processing the Mrna Transcript after Dna to Rna

Transcription

Capping- A cap is added to the 5 end and a poly-A tail (150 to 200 Adenines) is added to
the 3end of the molecule.
The introns are removed from the mRNA transcript.The remaining portions of mRNA are
called exons. They are spliced together to form a mature mRNA transcript.

Eukaryotic Transcription
In eukaryotes, transcription is achieved by three different types of RNA Polymerases. Rest of the
steps are same as prokaryotic transcription.

RNA pol I -this transcribes ribosomal RNAs (rRNAs)

RNA pol II - this transcribes messenger RNAs (mRNAs) and also small regulatory RNAs

RNA pol III- this transcribes transfer RNAs (tRNAs).

RNA pol II binds to a DNA sequence within the promoter of many genes, known as the TATA
box, to initiate transcription

The process of translation, involves sequential assembly of amino acids linked by a peptide bond
on an m-RNA which is bound by the ribosome complex. This is also known as decoding. The
process of translation involves three stages namely initiation, chain elongation and chain
termination. Protein synthesis is similar in both prokaryotes and eukaryotes, however minor
differences exist. The translational process described here is relevant to man and all other
eukaryotes.
Chain initiation:

The first step in translation is the attachment of the small 40S ribosome to the mRNA molecule.
An eukaryotic mRNA does not have an internal ribosome binding site, instead, the small subunit
of the ribosome recognizes the cap structure as its binding site and therefore attaches to the
extreme 5 end of the mRNA.
The translational process starts when an aminoacylated tRNA base pairs with an initiation codon
that has been locates by the small subunit of the ribosome. The initiator tRNA is charged with
methionine, because methionine is the amino acid coded by the AUG. The initiation process
involves ordered formation of a complex which constitute an initiator factor (eIF2), GTP, the
initiating methionine tRNA and the small 40S ribosome unit. This complex along with other
initiation factors bind to 5 end of mRNA and then moves along the mRNA until it reaches the
first AUG codon.

Translation:chain Elongation:
Once the initiation complex has formed the large subunit joins the complex, which requires the
hydrolysis of GTP and release of initiation factors. Two separate and distinct sites are now
available to which the tRNAs can now bind. The first of these called the peptidyl or P site is
occupied by the aminoacylated tRNA met which is still base paired with the initiation codon. The
aminoacyl or the A Site is positioned over the gene and at first it is empty. Elongation begins
when the correct aminoacylated tRNA enters the A site and base pairs with the second codon.
This requires the elongation factor eIF1, and a second molecule of GTP is hydrolyzed. Now both
the amino acids are in close contact and a peptide bond is formed which is catalyses by the
enzyme peptidyl transferase. Translocation occurs when the ribosome slips along the mRNA by
three nucleotides so the aa-aa-tRNA enters P site expelling the now uncharged tRNA met and
making the A site vacant again. The third aminoacylated tRNA enters the A site and the
elongation cycle is repeated.

Translation:chain Termination:
Termination of translation begins when a termination codon (UAA, UGA, UAG) enters the A
site. There are no tRNA molecules with anti codons able to base pair with any of the termination
codons. A termination factor eRF recognizes all the termination codons in a eukaryotes and it
requires the hydrolysis of GTP. The ribosome releases the polypeptide and mRNA and then
dissociates into subunits.