DNA Replication

DNA replication is a biological process that occurs in all living organisms and copies
their DNA; it is the basis for biological inheritance. The process starts when one double-
stranded DNA molecule produces two identical copies of the molecule. The cell cycle
(mitosis) also pertains to the DNA replication/reproduction process. The cell cycle
includes interphase, prophase, metaphase, anaphase, and telophase. Each strand of
the original double-stranded DNA molecule serves as template for the production of the
complementary strand, a process referred to as semiconservative replication.
Cellular proofreading and error toe-checking mechanisms ensure near perfect fidelity for
DNA replication.[1][2]

In a cell, DNA replication begins at specific locations in the genome, called

"origins".[3] Unwinding of DNA at the origin, and synthesis of new strands, forms
a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new
DNA by adding nucleotides matched to the template strand, a number of
other proteins are associated with the fork and assist in the initiation and continuation of
DNA synthesis.

DNA replication can also be performed in vitro (artificially, outside a cell). DNA
polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA
synthesis at known sequences in a template molecule. The polymerase chain
reaction (PCR), a common laboratory technique, employs such artificial synthesis in a
cyclic manner to amplify a specific target DNA fragment from a pool of DNA.
 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 templates

Leading strand The leading strand is the template strand of the DNA double helix so
that the replication fork moves along it in the 3' to 5' direction. This allows the newly
synthesized strand complementary to the original strand to be synthesized 5' to 3' in the
same direction as the movement of the replication fork.

On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it
continuously. This polymerase is DNA polymerase III (DNA Pol III) in prokaryotes and
presumably Pol ε[7][12] in yeasts. In human cells the leading and lagging strands are
synthesized by Pol α and Pol δ within the nucleus and Pol γ in the mitochondria. Pol ε
can substitute for Pol δ in special circumstances.[13]

Lagging strand The lagging strand is the strand of the template DNA double helix that is
oriented so that the replication fork moves along it in a 5' to 3' manner. Because of its
orientation, opposite to the working orientation of DNA polymerase III, which moves on
a template in a 3' to 5' manner, replication of the lagging strand is more complicated
than that of the leading strand.

On the lagging strand, primase "reads" the DNA and adds RNA to it in short, separated
segments. In eukaryotes, primase is intrinsic to Pol α.[14] DNA polymerase III or Pol
δ lengthens the primed segments, forming Okazaki fragments. Primer removal in
eukaryotes is also performed by Pol δ.[15] In prokaryotes, DNA polymerase I "reads"
the fragments, removes the RNA using its flap endonuclease domain (RNA primers are
removed by 5'-3' exonuclease activity of polymerase I [weaver, 2005], and replaces the
RNA nucleotides with DNA nucleotides (this is necessary because RNA and DNA use
slightly different kinds of nucleotides). DNA ligase joins the fragments together.

Transcription Processes

Transcription is the process of creating a complementary RNA copy of a sequence

of DNA.[1] Both RNA and DNA are nucleic acids, which use base
pairs of nucleotides as a complementary language that can be converted back and forth
from DNA to RNA by the action of the correct enzymes. During transcription, a DNA
sequence is read by an RNA polymerase, which produces a
complementary, antiparallel RNA strand. As opposed to DNA replication, transcription
results in an RNA complement that includes uracil (U) in all instances where thymine (T)
would have occurred in a DNA complement. Also unlike DNA replication where DNA is
synthesised, transcription does not involve an RNA primer to initiate RNA synthesis.

Transcription is explained easily in 4 or 5 steps, each moving like a wave along the
DNA.

1. RNA polymerase moves the transcription bubble, a stretch of unpaired

nucleotides, by breaking the hydrogen bonds between complementary
nucleotides.
2. RNA polymerase adds matching RNA nucleotides that are paired with
complementary DNA bases.
3. RNA sugar-phosphate backbone forms with assistance from RNA polymerase.
4. Hydrogen bonds of the untwisted RNA + DNA helix break, freeing the newly
synthesized RNA strand.
5. If the cell has a nucleus, the RNA is further processed (addition of a 3' poly-A tail
and a 5' cap) and exits through to the cytoplasm through the nuclear
pore complex.

Transcription is the first step leading to gene expression. The stretch of DNA
transcribed into an RNA molecule is called a transcription unit and encodes at least
one gene. If the gene transcribed encodes a protein, the result of transcription
is messenger RNA (mRNA), which will then be used to create that protein via the
process of translation. Alternatively, the transcribed gene may encode for either non-
coding RNA genes (such as microRNA, lincRNA, etc.) or ribosomal RNA (rRNA)
or transfer RNA (tRNA), other components of the protein-assembly process, or
other ribozymes.[2]

A DNA transcription unit encoding for a protein contains not only the sequence that will
eventually be directly translated into the protein (the coding sequence) but
also regulatory sequences that direct and regulate the synthesis of that protein. The
regulatory sequence before (upstream from) the coding sequence is called the five
prime untranslated region (5'UTR), and the sequence following (downstream from) the
coding sequence is called the three prime untranslated region (3'UTR).[2]

Transcription has some proofreading mechanisms, but they are fewer and less effective
than the controls for copying DNA; therefore, transcription has a lower copying fidelity
than DNA replication.[3]

As in DNA replication, DNA is read from 3' → 5' during transcription. Meanwhile, the
complementary RNA is created from the 5' → 3' direction. This means its 5' end is
created first in base pairing. Although DNA is arranged as two antiparallel strands in
a double helix, only one of the two DNA strands, called the template strand, is used for
transcription. This is because RNA is only single-stranded, as opposed to double-
stranded DNA. The other DNA strand is called the coding (lagging) strand, because its
sequence is the same as the newly created RNA transcript (except for the substitution
of uracil for thymine). The use of only the 3' → 5' strand eliminates the need for
the Okazaki fragments seen in DNA replication.[2]

Transcription is divided into 5 stages: pre-initiation, initiation, promoter

clearance, elongation and termination.

Protein Synthesis, TRANSLATION

In molecular biology and genetics, translation is the third stage of protein
biosynthesis (part of the overall process of gene expression). In translation, messenger
RNA (mRNA) produced by transcription is decoded by the ribosome to produce a
specific amino acid chain, or polypeptide, that will later fold into an active protein. In
Bacteria, translation occurs in the cell's cytoplasm, where the large and small subunits
of the ribosome are located, and bind to the mRNA. In Eukaryotes, translation occurs
across the membrane of the endoplasmic reticulum in a process called vectorial
synthesis. The ribosome facilitates decoding by inducing the binding
of tRNAs with complementary anticodon sequences to that of the mRNA. The tRNAs
carry specific amino acids that are chained together into a polypeptide as the mRNA
passes through and is "read" by the ribosome in a fashion reminiscent to that of a stock
ticker and ticker tape.

In many instances, the entire ribosome/mRNA complex causing it to bind to the outer
membrane of the rough endoplasmic reticulum and release the nascent protein
polypeptide inside for later vesicle transport and secretion outside of the cell. Many
types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear
RNA, do not undergo translation into proteins.

Translation proceeds in four

phases: initiation, elongation, translocation and termination (all describing the
growth of the amino acid chain, or polypeptide that is the product of translation). Amino
acids are brought to ribosomes and assembled into proteins.

In activation, the correct amino acid is covalently bonded to the correct transfer RNA
(tRNA). The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by
an ester bond. When the tRNA has an amino acid linked to it, it is termed "charged".
Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with
the help of initiation factors (IF). Termination of the polypeptide happens when the A site
of the ribosome faces a stop codon (UAA, UAG, or UGA). No tRNA can recognize or
bind to this codon. Instead, the stop codon induces the binding of a release
factor protein that prompts the disassembly of the entire ribosome/mRNA complex.

A number of antibiotics act by inhibiting translation; these

include anisomycin, cycloheximide, chloramphenicol, tetracycline, streptomycin, erythro
mycin, and puromycin, among others. Prokaryotic ribosomes have a different structure
from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial
infections without any detriment to a eukaryotic host's cells.
he central dogma of molecular biology describes the way genetic information is expected to

be transferred in a single direction through a biological system. It was first stated by Francis

Crick in 1958[1] and re-stated in a Nature paper published in 1970:[2]

The central dogma of molecular biology deals with the detailed residue-by-residue

transfer of sequential information. It states that such information cannot be transferred back

from protein to either protein or nucleic acid. Or, as Marshall Nirenberg said, "DNA makes RNA

makes protein."[3]

The dogma is a framework for understanding the transfer of sequenceinformation between

sequential information-carrying biopolymers, in the most common or general case, in

living organisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic

acids), and protein. There are 3×3 = 9 conceivable direct transfers of information that can occur

between these. The dogma classes these into 3 groups of 3: 3 general transfers (believed to

occur normally in most cells), 3 special transfers (known to occur, but only under specific

conditions in case of some viruses or in a laboratory), and 3 unknown transfers (believed

never to occur). The general transfers describe the normal flow of biological information: DNA

can be copied to DNA (DNA replication), DNA information can be copied

into mRNA (transcription), and proteins can be synthesized using the information in mRNA as a

template (translation).

Recombinant DNA (rDNA) molecules are DNA sequences that result from the
use of laboratory methods (molecular cloning) to bring together genetic material from multiple

sources, creating sequences that would not otherwise be found in biological organisms.

Recombinant DNA is possible because DNA molecules from all organisms share the same

chemical structure; they differ only in the sequence of nucleotides within that identical overall

structure. Consequently, when DNA from a foreign source is linked to host sequences that can

drive DNA replication and then introduced into a host organism, the foreign DNA is replicated

along with the host DNA.

Recombinant DNA molecules are sometimes called chimeric DNA, because they are usually

made of material from two different species, like the mythical chimera. R-DNA technology uses

palindromic sequences and leads to the production of sticky and blunt ends.

The DNA sequences used in the construction of recombinant DNA molecules can originate from

any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be

joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may

be created by the chemical synthesis of DNA, and incorporated into recombinant molecules.

Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be

created and introduced into any of a very wide range of living organisms.

Proteins that result from the expression of recombinant DNA within living cells are

termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a

host organism, the recombinant protein will not necessarily be produced.[citation needed]

Expression of foreign proteins requires the use of specialized expression vectors and often

necessitates significant restructuring of the foreign coding sequence.[citation needed]

Recombinant DNA differs from genetic recombination in that the former results from artificial

methods in the test tube, while the latter is a normal biological process that results in the

remixing of existing DNA sequences in essentially all organisms.

Molecular cloning is the laboratory process used to create recombinant DNA.[1][2][3][4] It is one

of two widely-used methods (along with polymerase chain reaction, abbr. PCR) used to direct

the replication of any specific DNA sequence chosen by the experimentalist. The fundamental

difference between the two methods is that molecular cloning involves replication of the DNA

within a living cell, while PCR replicates DNA in the test tube, free of living cells.

Formation of recombinant DNA requires a cloning vector, a DNA molecule that will replicate

within a living cell. Vectors are generally derived from plasmids or viruses, and represent

relatively small segments of DNA that contain necessary genetic signals for replication, as well

as additional elements for convenience in inserting foreign DNA, identifying cells that contain

recombinant DNA, and, where appropriate, expressing the foreign DNA. The choice of vector for

molecular cloning depends on the choice of host organism, the size of the DNA to be cloned,

and whether and how the foreign DNA is to be expressed.[5] The DNA segments can be

combined by using a variety of methods, such as restriction enzyme/ligase cloning or Gibson

assembly.

In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps:

(1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation

of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into

the host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for

clones with desired DNA inserts and biological properties.[4] These steps are described in some

detail in a related article (molecular cloning).

The Biosynthesis of Purines
.Figure 27.1  Nitrogen waste is excreted by birds principally as the purine analog, uric acid.

Substantial insight into the de novo pathway for purine biosynthesis was
provided in 1948 by John Buchanan, who cleverly exploited the fact that
birds excrete excess nitrogen principally in the form of uric acid, a water-insoluble
purine analog. Buchanan fed isotopically labeled compounds to pigeons and then
examined the distribution of the labeled atoms in uric acid (Figure 27.1). By tracing
the metabolic source of the various atoms in this end product, he showed that the nine
atoms of the purine ring system (Figure 27.2) are contributed by aspartic acid (N-1),
glutamine (N-3 and N-9), glycine (C-4, C-5, and N-7), CO2 (C-6), and THF one-
carbon derivatives (C-2 and C-8). The coenzyme THF and
its role in one-carbon metabolism were introduced
in Chapter 18.

Figure 27.2  The metabolic origin of the nine atoms in the purine ring system
Regulation of Pyrimidine Biosynthesis

Pyrimidine biosynthesis in bacteria is allosterically regulated at aspartate trans-

carbamoylase (ATCase). Escherichia coli ATCase is feedback-inhibited by the end
product, CTP. ATP, which can be viewed as a signal of both energy availability and
purine sufficiency, is an allosteric activator of ATCase. CTP and ATP compete for a
common allosteric site on the enzyme. In many bacteria, UTP, not CTP, acts as the
ATCase feedback inhibitor.
In animals, CPS-II catalyzes the committed step in pyrimidine synthesis and
serves as the focal point for allosteric regulation. UDP and UTP are feedback
inhibitors of CPS-II, while PRPP and ATP are allosteric activators. With the
exception of ATP, none of these compounds are substrates of CPS-II or of either of
the two other enzymic activities residing with it on the trifunctional polypeptide.
Figure 27.19 compares the regulatory circuits governing pyrimidine synthesis in
bacteria and animals.