Mol Bio
Johann Friedrich Miescher
CHAPTER 1
James Watson
 coined the term molecular biology,1 he was referring to
the biology of deoxyribonucleic acid (DNA).
DEOXYRIBONUCLEIC ACID (DNA)
 It is a Macromolecule: carbon, nitrogen, oxygen,
phosphorous, and hydrogen
 Assembled in Nucleotides: phosphorylated ribose
sugar and a nitrogen base
 Four nitrogen bases : adenine, cytosine, guanine,
and thymine
o NB= attached to deoxyribose sugar, which
forms a polymer with the deoxyribose sugar
of other nucleotides through
phosphodiester bond.
 Linear assembly of nucleotides = one strand of
DNA
 DNA (Deoxyribonucleic acid)
 It is a human hereditary material
 it is a carrier of all genetic information
 DNA’s structure is a double-stranded helix,
and it resembles the look of a twisted ladder.
 Long molecule containing the information
organisms need to develop and reproduce
 It is found in every cell in the body and
passed down from parent to child
 DNA replicates on its own, it is self-
replicating.
 most DNA is found in the nucleus of a cell, a
small amount can also be found in the
mitochondria, which generates energy so
cells can function properly. Perhaps the
most fascinating part of the process is the
fact that nearly every cell in your body has
the same DNA.
 A nucleotide is the basic structural unit and building
block for DNA. These building blocks are hooked
together to form a chain of DNA. A nucleotide is
composed of 3 parts:
 five-sided sugar
 phosphate group
 nitrogenous base (nitrogen containing)
 The bases are located in the middle of the DNA
double helix, while the sugar and phosphate groups
form the backbone. The backbone is held together by
a chemical bond formed between the phosphate
group of one nucleotide and the sugar of a
neighboring nucleotide. The two strands of the double
helix are held together by chemical bonds (hydrogen
bonds) formed between opposing bases.
 There are four types of bases in DNA. They are called:
 Adenine (A)
 Cytosine (C)
 Guanine (G)
 Thymine (T)
o In RNA, the base uracil (U) takes
the place of thymine.
 Bases are the part of DNA that stores information and
gives DNA the ability to encode phenotype, a
person’s visible traits.
 Adenine and guanine are purine bases. These are
structures composed of a 5-sided and 6-sided ring.
 Cytosine and thymine are pyrimidines which are
structures composed of a single six-sided ring.
Adenine always binds to thymine, while cytosine and
guanine always bind to one another.
 This relationship is called complementary base
paring. These complementary bases are bonded
together via hydrogen bonds, which can be easily
broken apart when the DNA needs to unzip and
duplicate itself.
 1871
 published a paper on nuclein, the viscous substance
extracted from cell nuclei
 In his writings, he made no mention of the function
of nuclein.
 1869
 Credited with discovery of DNA
 He isolated it from white blood cells found in
discarded surgical bandages.
 The substance was found in cell nuclei and could be
extracted with dilute hydrochloric acid.
 Addition of pig stomach extract removed
contaminating proteins, leaving clean nuclei.
 Alkali extraction yielded the same substance found
in intact cells.
 Chemical analysis showed nuclein was 14%
nitrogen and 2.5% phosphorus.
 Less than 30% of Miescher's first nuclein
preparation was actually DNA.
 Miescher also isolated a similar substance from
salmon sperm, suggesting a role in fertilization.
WALTER FLEMMING
 a leading cell biologist, describing his work on the nucleus
in 1882 admitted that the biological significance of the
substance was unknown
DNA STRUCTURE
 James Watson and Francis Crick first described the
double helical structure of DNA.
 Their model was based on previous observations of
DNA's chemical nature and diffraction analyses by
Rosalind Franklin.
 The helical structure arises from the linear
arrangement of nucleotides.
 Specific nucleotide sequences and the surrounding
chemical environment influence the DNA helix.
 Two regions: (form on the opposite sides of the base
pairs or cut that is present in the structure of DNA)
o Major groove (5’)
- takes place where the sugar
phosphate backbones are close
together.
- wider than the minor groove
(many proteins which bind to B-
DNA do so through the wider major
groove)
o Minor groove (3’)
- takes place where the sugar
phosphate backbones are far apart.
 Double stranded in nature.
 DNA’s structure is a double-stranded helix,
and it resembles the look of a twisted ladder.
 The double helix of DNA has sugar-phosphate backbones
arranged at specific distances from each other.
 Two regions formed by the backbones are the major
groove and minor groove.
 Proteins bind to specific nucleotide sequences in DNA at
these grooves (binding or recognition sites).
 Intercalating agents can slide transversely into the center
of the helix.
 Denaturing agents like formamide and urea displace
hydrogen bonds, separating the two strands of the helix.
 B-form
o standard and hydrated form of DNA.
o 10.5 pairs of nucleotides (base pairs [bp])
per turn.
 is a canonical DNA helix and the most
common type of DNA.
  B-form DNA is a right-handed double helix,
which was discovered by Watson and Crick
based on the X-ray diffraction patterns.
 A-form
o dehydrated DNA with 11 base pair per turn;
center of symmetry along the outside of the
helix rather than down the middle as it is in
B-form.
 is an active double helix structure with a
shorter and more complicated DNA form
 A-DNA is a right-handed double helix made
up of deoxyribonucleotides.
 The two strands of A-DNA are anti-parallel
with each other and not symmetrical.
 Both A- and B- form DNA are right-handed helices.
 Z-DNA
o formed when stress and torsion is applied.
o Left-handed helix with 12 base per per turn;
altered geometry of the sugar-base bonds.
 is left-handed double helix DNA with a
zigzag pattern.
 Z-form DNA is a left-handed double helix. It
has a very different structure when
compared with A-DNA and B-DNA. The
zigzag appearance of backbone allows it to
be distinguished from other forms of DNA.
 Same as the other forms of DNA, hydrogen
bond is present to hold the two strands
together.
 Z-DNA is difficult to be observed since it is
unstable. It may take part in expression
regulation of some genes or in genetic
recombination.
 It can be found in bacteria, eukaryotes and
viruses. In some viruses, they require Z-DNA
binding proteins for pathogenesis.
EXAMPLE PIC:
 Watson and Crick's described DNA's standard form as
the hydrated B-form, with 10.5 base pairs per turn.
 Dehydrated DNA takes the A-form, with about 11 base
pairs per turn and a center of symmetry along the
outside of the helix.
 Both A- and B-form DNA are right-handed helices.
 Stress and torsion can cause DNA to adopt the left-
handed Z-form, with 12 base pairs per turn and altered
sugar-base bond geometry.
 Watson-Crick base pairing is not exclusive to DNA and
RNA; alternatives like the pentopyranosyl-(2'→4')
oligonucleotide system exist.
 Nucleic acid alternatives have practical applications such
as protein nucleic acids, which can be used in the
laboratory as hybridization probes and in antisense RNA
therapies.
 Hydrogen bonds
o are key to the specificity of most nucleic
acid-based test used in molecular lab.
 Hydrogen bonds are found between the two strands
of DNA where the bases pair with each other.
 Adenine (A) pairs with thymine (T), and guanine
(G) pairs with cytosine (C).
 Two hydrogen bonds form between A and T, and
three hydrogen bonds form between G and C.
 The GC base pair has a stronger bond than the AT
base pair because of the extra hydrogen bond.
 Each DNA molecule consists of two nucleotide chains
wrapped around each other in a double helix and held
together by hydrogen bond
 hydrogen bonding involves only the
nitrogenous bases
 Each of the purine bases can hydrogen bond with one
and only one of the pyrimidine bases
 They are known to play a key role in the maintenance
of nucleic acid and protein three-dimensional
structures.
NUCLEOTIDES
 DNA's nucleotide building blocks are about 700 kd in size.
 Each nucleotide comprises a five-carbon sugar, a
nitrogen base, and a phosphate moiety.
 Nucleosides consist of a nitrogen base bound to an
unphosphorylated sugar.
 Phosphorylation of the ribose sugar creates nucleoside
mono-, di-, or triphosphates, or nucleotides.
 Adenosine monophosphate (AMP) and adenosine
triphosphate (ATP) are examples of nucleotides.
 Free nucleotides in DNA are deoxyribonucleoside
triphosphates (e.g., dATP).
 DNA's sugar is deoxyribose, lacking a hydroxyl group on
its second carbon.
 Nitrogen bases in DNA are planar carbon-nitrogen ring
structures.
 The four common nitrogen bases in DNA are adenine,
guanine, cytosine, and thymine.
 Thymine and cytosine are pyrimidines, while guanine and
adenine are purines.
 Numbering of positions in nucleotides starts with the
nitrogen base ring positions (C or N1, 2, 3, etc.).
 The carbons of the ribose sugar are numbered 1' to 5',
distinguishing them from the nitrogen base ring positions.
 A nucleotide is an organic molecule that serves as the
foundation for DNA and RNA.
 There are four nucleotides, or bases, in
DNA: adenine (A), cytosine (C), guanine (G), and
thymine (T). These bases form specific pairs (A with
T, and G with C).
 A molecule consisting of a nitrogen-containing base
(adenine, guanine, thymine, or cytosine in DNA;
adenine, guanine, uracil, or cytosine in RNA), a
phosphate group, and a sugar (deoxyribose in DNA;
ribose in RNA).
 DNA chains form hydrogen bonds between
complementary bases: guanine pairs with cytosine (3
hydrogen bonds), adenine pairs with thymine (2 hydrogen
bonds).
 Specific hydrogen bond formation maintains the linear
order of nucleotides, crucial for information preservation
during DNA replication.
 DNA replication involves complementary base pairing
(A:T, G:C) between parental and newly synthesized DNA
strands.
 Base pairs other than A:T and G:C, or mismatches, can
distort the DNA helix and disrupt sequence
information.
 Drugs like AZT, ddC, ddI, and Acyclovir are
modifications of nucleosides used in treating HIV, herpes
simplex virus, and varicella-zoster virus.
 In the laboratory, nucleosides can be modified for
labeling, detection, sequencing, and other applications.
 Nucleoside
o nitrogen base bound to an unphosphorylated
sugar
 a compound (such as guanosine or adenosine)
composed of a purine or pyrimidine base combined
 with deoxyribose or ribose and found primarily in DNA
or RNA
Purines and Pyrimidines are nitrogenous bases that
make up the two different kinds of nucleotide bases
in DNA and RNA.
The two-carbon nitrogen ring bases (adenine and
guanine) are purines, while the one-carbon nitrogen
ring bases (thymine and cytosine) are pyrimidines.
 Pyrimidines
o Nitrogen bases with a single-ring structure
(thymine, cytosine)
 Single carbon-nitrogen ring with two nitrogen atoms
 Cytosine in both DNA and RNA
 Uracil only in RNA
 Thymine only in DNA
 Purines
o bases with a double-ring structure (guanine,
adenine)
 Double carbon-nitrogen ring with four nitrogen atoms
 Adenine and Guanine in both DNA and RNA
Guanosine monophosphate (dGMP)
Guanosine monophosphate (GMP) is a nucleoside phosphate
with one phosphate group and one ribonucleoside group. It has
ribose as its sugar and one phosphate group attached to it. Its
nucleoside (guanosine) is composed of a purine base, i.e.
guanine, linked to ribose sugar. The nucleoside has only one
phosphate group attached to it.
MODIFIED NUCLEOSIDES
NUCLEOTIDES
 Sequences of the two strands are complementary, not
identical.
 Antiparallel orientation orientation, with the 5 ′ end of
one strand at the 3 ′ end of the other
 The strands are anti-parallel, which means they fit
together by facing opposite directions.
 Hybridization
o formation of hydrogen bonds between two
complementary strands of DNA.
 Adenine (A) pairs with thymine (T),
 guanine (G) pairs with cytosine (C).
During DNA synthesis, the new strand is built by adding
nucleotides from the 5' phosphate end to the 3' hydroxyl end.
Since the template strand is read in the opposite direction (3' to
5'), the new strand is synthesized in the opposite direction. As
a result, the new double helix consists of one strand running in
the 5' to 3' direction (the new daughter strand) and the other
running in the 3' to 5' direction (the template strand).
DNA REPLICATION
 DNA strands in a double helix are antiparallel due to
replication.
 DNA synthesis occurs from 5' to 3', while the enzyme
DNA polymerase reads the template in the 3' to 5'
direction.
 This results in one strand serving as the parent template
and the other being newly synthesized in the opposite
orientation.
 As Watson and Crick predicted, semi-conservative
replication is the key to maintaining the sequence of the
nucleotides in DNA through new generations.
 Semi-conservative replication ensures faithful
transfer of DNA sequence information.
 The replication apparatus is designed to copy DNA with
minimal errors before each cell division.
 DNA replication begins with unwinding the double helix.
 Each single strand serves as a template for new strand
synthesis.
 The new strand elongates through complementary base
pairing and nucleophilic attack.
 A phosphodiester bond forms between the new
nucleotide and the last nucleotide of the growing chain.
 The duplicated helix consists of one template strand and
one newly synthesized strand.
 DNA replication proceeds through both strands of the
DNA duplex simultaneously.
 Replication forks, observed by electron microscopy,
indicate active replication.
 Okazaki and Okazaki's experiments revealed small
DNA fragments, about 1,000 bases long, formed during
replication.
 These fragments, termed Okazaki fragments, are
covalently linked shortly after synthesis.
 Okazaki fragments explain how both strands are
copied at the replication fork.
 The leading strand is copied continuously from 3' to 5',
while the lagging strand is copied discontinuously.
 Primase is an RNA-synthesizing enzyme that catalyzes
the synthesis of short (6 to 11 bp) RNA primers required
for priming DNA synthesis.
 Primase must work repeatedly on the lagging strand
to prime synthesis of each Okazaki fragment
 DNA replication is the process by which DNA duplicates
itself during cell division.
1. The first step in DNA replication is to ‘unzip’ the
double helix structure of the DNA? molecule.
2. This is carried out by an enzyme? called helicase
which breaks the hydrogen bonds? holding
the complementary? bases? of DNA together (A with T,
C with G).
3. The separation of the two single strands of DNA
creates a ‘Y’ shape called a replication ‘fork’. The two
separated strands will act as templates for making the
new strands of DNA.
4. One of the strands is oriented in the 3’ to 5’ direction
(towards the replication fork), this is the leading
strand?. The other strand is oriented in the 5’ to 3’
direction (away from the replication fork), this is
the lagging strand?
 DNA polymerase
o enzyme responsible for polymerizing the
nucleotide chains.
 are enzymes that assemble nucleotides, the building
blocks of DNA, to form DNA molecules.
 Template
o a guide, determine which nucleotides to add
to the chain
 It serves as a guide for determining which nucleotides
to add to the chain.
 refers to the DNA sequence that has the ability to
replicate itself during mRNA synthesis.
 Semi-conservative replication (Watson and Crick
predicted,)
o key to maintaining the sequence of the
nucleotides in DNA through new generations.
  DNA replication is a semi-conservative process
because when a new double-stranded DNA molecule
is formed, one strand will be from the original
template molecule. One strand will be newly
synthesised.
 According to the semiconservative replication model,
during replication, the two original DNA strands (i.e.,
the two complementary halves of the double helix)
separate; each strand then serves as a template for a
new DNA strand, which means that each newly
synthesized double helix is a combination of one old
(or original) and one new DNA strand.
Erwin Chargaff
 observed that the amounts of adenine in DNA equaled
the amounts of thymine, and the amounts of cytosine
equaled the amounts of guanine.
James Watson
 proposed that the steps in the double helix ladder were
pairs of bases: thymine with adenine, and guanine with
cytosine.
James Watson and Francis Crick
 suggested that this base pairing arrangement formed the
basis for a DNA copying mechanism.
 Complementary DNA strands could separate and act as
templates for producing new complementary strands.
Matthew Meselson and Franklin Stahl
 demonstrated semi-conservative replication a few years
after the double helix solution.
 They used equilibrium density centrifugation on a cesium
gradient
 Heavy DNA was prepared by growing bacteria in a
medium containing the nitrogen isotope 15N.
 Shifting the bacteria to normal nitrogen (14N) medium
allowed separation of hybrid 14N:15N DNA molecules.
 These molecules had a specific intermediate density
compared to those from bacteria grown only in 14N or
15N.
 True semi-conservative replication was differentiated from
dispersive replication by showing approximately half of
the DNA double helices from the next generation were
14N:15N and half were 14N:14N.
NUCLEIC ACID
 Nucleic acids are macromolecules composed of
nucleotides linked by phosphate and hydroxyl
groups on their sugars.
 Nucleic acid chains grow as the 5' phosphate group of
an incoming nucleotide attaches to the 3' hydroxyl
group of the last nucleotide.
 This attachment creates a polarity in the DNA chain,
with a 5' phosphate end and a 3' hydroxyl end.
 DNA is oriented in a 5' to 3' direction, and the linear
sequence of nucleotides is conventionally read in that
order.
PROCESS OF REPLICATION
DNA replication is a template-guided polymerization that DNA
polymerase catalyzes. The new thread is
Synthesized in the 5 ′ to 3 ′ direction, with the template strand
read in the same direction.
Both strands of the double helix are replicated at the same
time. Both strands are read in the direction of 3 ′ to 5 ′. The
polymerase skips ahead and reads back toward the replication
fork, reading one strand oriented 5 ′ to 3 ′ (the lagging strand).
 Primase is an enzyme that creates primers, which
are short RNA sequences that serve as starting points
for DNA synthesis.
 DNA helicases are required during DNA replication
because they split double-stranded DNA into single
strands, allowing each strand to be copied
independently.
 Rnase H hydrolyzes the phosphodiester bonds of
RNA, when hybridized to DNA.
POLYMERASES
 Enzymes that synthesizes long chains of polymers or
nucleic acid.
 DNA polymerase I (pol I) was the first purified
enzyme shown to catalyze DNA replication in
prokaryotes.
 DNA polymerases II (pol II) and III (pol III) were
later characterized, with pol III identified as the main
polymerizing enzyme during bacterial replication.
 Pol I, II, and III are responsible for repair of gaps and
discontinuities in previously synthesized DNA.
 Pol I was preferentially purified in early studies due to
its higher activity compared to pol II and pol III.
 Pol III functions as a multi-subunit holoenzyme in vivo,
working alongside other proteins involved in
replication initiation, regulation, and termination.
 The pol III holoenzyme includes two catalytic DNA
polymerizing enzymes—one for the leading strand
synthesis and one for the lagging strand synthesis.
 DNA polymerase I (pol I)
o first purified enzyme to catalyze DNA
replication in prokaryotes
 DNA polymerase 1 is the main enzyme for repair,
removal of primers and filling the gaps in the lagging
strand.
 DNA polymerase III (pol III)
o main polymerizing enzyme during bacterial
replication
 DNA polymerase 3 is the main enzyme catalysing the
5'→3' polymerisation of DNA strand during replication
 Holoenzymes
o required for priming, initiation, regulation,
and termination of the replication process.
 Holoenzyme is a biochemically active compound
formed by the combination of an enzyme with a
coenzyme.
 Common examples of holoenzymes are: RNA
polymerase, DNA polymerase, Carbonic
anhydrase, Haemoglobin, Cytochrome
oxidase, Pyruvate carboxylase
  The reaction required preformed DNA, all four nucleotides,
and a bacterial protein extract.
 Any source of preformed DNA—bacterial, viral, or
animal—could be used.
 Initially, it was unclear whether the new DNA was a copy
or an extension of the input molecule.

Julius Adler, Sylvy Kornberg, and Steven B.

Zimmerman
 showed within 3 years that the new DNA had the same A-
T to G-C base pair ratio as the input DNA, confirming it
was indeed a copy.
 The ratio was unaffected by the proportion of free
nucleotides added, indicating that the input or template
DNA determined the sequence of the newly synthesized
DNA.

-ADVANCE CONCEPT
 Polymerase complexes α and β are found in the nucleus,
while γ is found in the mitochondria.
 Polymerases α, β, and γ resemble prokaryotic enzymes
but have less demonstrable exonuclease activity.
 Polymerase δ, originally isolated from bone marrow,
possesses 3' to 5' exonuclease activity.
 Polymerase α is primarily involved in chromosome
replication, while β and δ are associated with DNA repair.

 After replication, distortions in DNA caused by

mismatched or aberrantly modified bases are removed by
the 5' to 3' exonuclease function of repair polymerases
like DNA pol I.
 This activity degrades duplex DNA from the 5' end and
can cleave diester bonds several bases from the end of
the chain.
 It is crucial for removing lesions such as thymine or
pyrimidine dimers induced by exposure to ultraviolet light.
 Failure to remove these lesions can disrupt subsequent
transcription and replication of the DNA strand.

DNA POLYMERASES
Multiple protein molecules are involved in DNA polymerase  Functions:
activity. To unwind the template helix, prime synthesis with A. Polymerization
RNA primers, and protect the lagging strand, a number of B. Pyrophosphorolysis
cofactors and accessory proteins are required. C. Pyrophosphate exchange
D. Synthesize DNA in a 5’ to 3’ direction
 Most DNA polymerases possess multiple functions, E. Degrade DNA in both a 5’ to 3’ and 3’ to 5’
including polymerization, pyrophosphorolysis, and direction
pyrophosphate exchange, allowing for both synthesis and
degradation of DNA. DNA POL I
 E. coli DNA pol I's catalytic domain has two fragments: a
large fragment with polymerase activity and a small
fragment with exonuclease activity.
 The exonuclease function serves to protect the DNA
sequence by proofreading newly synthesized DNA,
correcting any misincorporated nucleotides.
 E. coli DNA pol III can simultaneously synthesize and
degrade DNA during replication through nick translation.
 Nick translation involves the enzyme adding nucleotides
at the 3' end of a nick while removing nucleotides ahead
of it with its 5' to 3' exonuclease function.
 DNA ligase subsequently reseals the nick, forming
phosphodiester bonds between existing DNA strands.  The Klenow fragment is a large protein fragment
 Nick translation is utilized in vitro to introduce labeled produced when DNA polymerase I from E. coli is
nucleotides into DNA molecules for detection in enzymatically cleaved by the protease subtilisin.
hybridization analyses.  Klenow fragment lacks 5′ to 3′ exonuclease activity

 Terminal transferase, a type of DNA polymerase, can  5´→3´ exonuclease activity, which enables the
synthesize polynucleotide chains without a template. enzyme to replace nucleotides in the growing strand
 This enzyme adds nucleotides to the end of a DNA of DNA by nick translation.
strand in the absence of hydrogen base pairing with
a template.  Exonucleases are enzymes that catalyze the
 Terminal transferase is used in the laboratory to removal of nucleotides from the ends of single-
generate 3'-end labeled DNA species. stranded and/or double-stranded DNA in either the 5-
 DNA polymerases are essential in biotechnology for prime to 3-prime or 3-prime to 5-prime directions.
cloning, amplification, and sequencing.
 Polymerases are classified into families (A, B, C, X) NICK TRANSLATION
based on sequence structure.
 Polymerases in the A and B family are most useful
for biotechnological engineering because they are
monomeric.
 Chemical manipulation of amino acid structure can
produce polymerases with altered characteristics useful in
the laboratory, such as altered processivity, fidelity, and
substrate specificity.

Arthur Kornberg, I. Robert Lehman, and Maurice J.

Bessman
 reported on an E. coli extract that could polymerize
nucleotides into DNA in vitro in 1956.
Nick translation is a tagging technique that involves using DNA
polymerase I to replace some of the nucleotides in a DNA
sequence with labeled analogues. DNA molecules are first
treated with DNase to create single-stranded "nicks" in this
process. Then, using 5'-3' exonuclease activity, DNA
polymerase I elongates the 3' hydroxyl terminus of the nicked
sites, removing nucleotides and replacing them with modified
dNTPs or deoxynucleotide triphosphate, labeling the DNA
molecule. This technique is commonly used to label probes in
fluorescence in situ hybridization (FISH).
TERMINAL TRANSFERASE
 One type of DNA polymerase
 Can synthesize polynucleotide chains without a
template
 Add nucleotides to the end of a DNA strand in the
absence of hydrogen base pairing with a template
 Used in laboratory to generate 3’-end labeled DNA
species
 Characteristics of polymerases:
1. Processivity
2. Fidelity
3. Substrate specificity
 TdT is a template independent polymerase that
catalyzes deoxynucleotide addition to the 3' hydroxyl
terminus of DNA molecules.
ENZYMES THAT METABOLIZE DNA
A. Restriction Enzymes
o are endonucleases that recognize specific
sequences and break or restrict the DNA
polymer at the sugar-phosphate backbone.
o Originally isolated from bacteria, where they
function as part of a primitive immune
system.
o They are named for the organism from which
they were isolated.
 Restriction enzymes recognize specific DNA
sequences and cut them in a controlled way.
B. DNA ligase
o catalyzes the formation of a phosphodiester bond
between adjacent 3’-hydroxyl and 5’-phosphoryl
nucleotide ends.
o DNA ligase works by catalyzing the formation of
a phosphodiester bond between nucleotides on one
strand of a double stranded DNA molecule.
C. Nucleases
o used, under controlled conditions, to manipulate DNA
in vitro.
o Nucleases are enzymes that degrade nucleic acids.
They play critical roles in genetic quality control, such
as DNA proofreading during replication, base,
nucleotide, mismatch, and double-strand repairs,
homologous recombination, and turnover.
 Exonucleases vs. Endonucleases:
 Exonucleases degrade DNA from free 3′-hydroxyl or
5′-phosphate ends, unlike endonucleases.
 Unsuitable for closed circular DNA due to their mode
of action.
 Applications:
 Used under controlled conditions for DNA
manipulation in vitro.
 Applied for making stepwise deletions in linearized
DNA or modifying DNA ends post-restriction enzyme
digestion.
 Types and Functions:
 Exonuclease I (from E. coli):
 Degrades single-stranded DNA from the 3′-
hydroxyl end into mononucleotides.
 Optimal activity on long single-stranded ends.
 Useful for removing excess primers from
double-stranded DNA products.
 Exonuclease III (from E. coli):
 Removes 5′ mononucleotides from the 3′ end
of double-stranded DNA in the presence of
Mg2+ and Mn2+.
 Can also act as an endonuclease at apurinic
sites.
 Effective on blunt ends, recessed ends, and
nicks but not on 3′ overhangs.
 DNA by separating the sugar-phosphate backbones.
 Exonuclease VII (from E. coli):
 Digests single-stranded DNA from either the 5′-
phosphate or 3′-hydroxyl end.
 Exhibits 5′ exonuclease activity, a rare characteristic.
 Used to remove long single strands from double-
stranded DNA or single strands from DNA mixtures.
 Nuclease Bal31 (from Alteromonas espejiani):
 Degrades single- and double-stranded DNA from
both ends.
 Can act as an endonuclease on single-stranded
DNA.
 Useful for making nested deletions in DNA due to its
controllable activity at 20°C.
 Mung Bean Nuclease (from Mung Bean Sprouts):
 Digests single-stranded DNA and RNA while
preserving double-stranded regions.
 Used to remove overhangs from restriction
fragments for producing blunt ends in cloning.
 Activity on RNA aids in transcriptional mapping and
resolving RNA hairpins.
 S1 Nuclease (from Aspergillus oryzae and certain
Neurospora species):
 Hydrolyzes single-stranded DNA or RNA into 5′
mononucleotides.
 Capable of endonuclease activity on single-stranded
regions in duplex DNA.
 Used in early nuclease mapping assays of DNA-
and RNA-binding proteins.
 recBC Nuclease (Exonuclease V) (from E. coli):
 ATP-dependent single- and double-stranded DNA
nuclease.
 Digests single-stranded DNA from 3′-hydroxyl or 5′-
phosphate ends.
 Exhibits endonuclease activity on duplex DNA and
can unwind the DNA double helix at high ATP levels.
 Micrococcal Nuclease:
 Digests single- and double-stranded DNA and RNA,
especially at AT- or AU-rich regions.
 Preference for single-stranded substrates, useful in
removing nucleic acids from crude extracts and
analyzing chromatin structure.
 Deoxyribonuclease I (DNAse I) (from bovine
pancreas):
 Digests single- and double-stranded DNA at
pyrimidines to oligonucleotides.
 Used to remove DNA from RNA preparations and
detect exposed DNA regions in DNA-protein binding
experiments.
 DNA Pol I (from E. coli):
 Has exonuclease activity, formerly known as
exonuclease II, responsible for proofreading during
polymerization.
 Nuclease Elimination:
 Critical to eliminate or inactivate nucleases during
nucleic acid specimen preparation for clinical
analysis.
 Most DNA isolation procedures minimize
endonuclease and exonuclease activity.
 Purified DNA often stored in TE buffer to chelate
cations required by nucleases.
D. Helicases
o carried out series of functions in DNA transcription,
replication, and recombination
o Helicases are enzymes that bind and may even
remodel nucleic acid or nucleic acid protein
complexes
o DNA helicases are essential during DNA replication
because they separate double-stranded DNA into
single strands allowing each strand to be copied.
 DNA Organization in Bacteria and Eukaryotes:
 DNA exists as a series of highly organized loops
and coils, not a relaxed double helix.
 Release of DNA for transcription, replication, and
recombination without tangling is facilitated by
cutting and re-closing of the DNA sugar-phosphate
backbone.
 Functions of Helicases:
 Enzymes called helicases facilitate the release of
  Double-strand breaks (DSBs) or single-strand
breaks (nicks) allow free rotation of broken ends
around intact strands.
 Helicases can digest the broken ends with
exonuclease activity or extend them using intact
strands as templates (nick translation).
 Helicases relieve topological stress in highly
compacted or supercoiled DNA, essential for
processes like DNA replication or transcription.
 Types of Helicases
 Topoisomerases:
 Interconvert topological isomers or relax
supercoiled DNA.
 Gyrases (Type II Topoisomerases):
 Untangle DNA through double-strand breaks
and separate linked rings of DNA
(concatemers).
 Role of Topoisomerases in Eukaryotes:
 Eukaryotic topoisomerases have similar activities to
those in bacteria but with different mechanisms of
cutting and binding to released DNA ends.
 Topoisomerases are crucial in cell replication and
are targets for anticancer drugs like camptothecin,
epipodophyllotoxins (VP-16 and VM-26), amsacrine,
doxorubicin, and mitoxantrone.
 Topoisomerase inhibitors induce cell death by
interfering with the breaking and joining activities of
the enzymes, trapping unfinished and broken
intermediates.
E. Methyltransferases
o catalyze the addition of methyl groups to nitrogen
bases, usually adenines and cytosines in DNA
strands.
 Once DNA is polymerized, it requires selective tapping to
make RNA while being protected from damage.
 Protective systems in prokaryotes eliminate foreign DNA,
such as infecting bacteriophages or viruses, using
enzymes that modify and digest foreign DNA.
 In sexually reproducing organisms, the mixing of parental
DNA generates genetic diversity in offspring, requiring
cutting and reassembly of DNA strands prior to cell
division and gamete formation.
 Enzymes perform these functions during various stages
of the cell cycle, including DNA polymerase, which has
been isolated for in vitro manipulation of DNA in the
laboratory.
 These enzymes are crucial tools in recombinant DNA
technology, forming the basis for commonly used
molecular techniques.
o An enzyme that attaches methyl groups to DNA (a
protein that speeds up chemical reactions in the body).
A methyl group is a chemical group composed of one
carbon atom and three hydrogen atoms. Also known
as DNA methylase.
 Function of DNA Methyltransferases:
 DNA methyltransferases catalyze the addition of
methyl groups to nitrogen bases, predominantly
adenines and cytosines in DNA strands.
 Prokaryotic DNA Methylation:
 Most prokaryotic DNA is methylated or
hemimethylated, meaning methylated on one strand
of the double helix and not the other.
 Prokaryotic DNA methylation serves to differentiate
host DNA from non-host DNA and provides
resistance to restriction enzymes.
 Eukaryotic DNA Methylation:
 In contrast to prokaryotes, eukaryotic DNA is
methylated in specific regions.
 DNA-binding proteins may limit accessibility to
certain regions or guide methyltransferases to
specific regions of the DNA in eukaryotes.
NUCLEIC ACID
 Nucleic acids are macromolecules composed of
nucleotides linked by phosphate and hydroxyl
groups on their sugars.
 Nucleic acid chains grow as the 5' phosphate group of
an incoming nucleotide attaches to the 3' hydroxyl
group of the last nucleotide.
 This attachment creates a polarity in the DNA chain,
with a 5' phosphate end and a 3' hydroxyl end.
 DNA is oriented in a 5' to 3' direction, and the linear
sequence of nucleotides is conventionally read in that
order.
RESTRICTION ENZYMES
- Endonucleases, such as restriction enzymes, break the
sugar-phosphate backbone of DNA and are crucial tools in
genetic engineering.
- Restriction enzymes recognize specific base sequences and
cleave DNA at the sugar-phosphate backbone, initially isolated
from bacteria where they function as part of an immune system
against foreign DNA.
- They are named based on the organism from which they
were isolated, and classified into four types (Type I, II, III, and
IV), with Type II being the most commonly used in the
laboratory.
Type I Restriction Enzymes
 Have both nuclease and methylase activities in a single
enzyme.
 Bind to host-specific DNA sites of 4 to 6 base pairs,
separated by 6 to 8 base pairs, containing methylated
adenines.
 Cleavage of the DNA substrate can occur over 1,000
base pairs from the binding site.
 Example: EcoK from E. coli K12.
Type II Restriction Enzymes
 Most frequently used in the laboratory.
 Do not have inherent methylation activity.
 Bind to symmetrical 4 to 8 base pair DNA recognition
sites, which are palindromic.
 Cleave DNA directly at the binding site, producing
fragments of predictable size.
 Their action forms the basis of many molecular
techniques, including mapping, cloning, genetic
engineering, and mutation analysis.
 Some enzymes produce staggered cuts, resulting in
sticky ends, while others produce blunt ends.
 Sticky ends facilitate DNA recombination by directing the
efficient joining of complementary ends.
 Example: EcoRI, HindIII.
**Applications:**
 Used in various molecular biology techniques such as
mapping, cloning, genetic engineering, and mutation
analysis.
 Important in medical laboratories for analyzing gene
rearrangements and mutation detection.
Type III Restriction Enzymes
 Resemble Type I enzymes in their ability to methylate and
restrict DNA.
 Have multiple subunits, including helicase activity.
 Recognition sites are asymmetrical, and cleavage of the
substrate DNA occurs 24 to 26 base pairs from the site to
the 3' side.
 Example: PstII from P. stuartii.
Type IV Restriction Enzymes
 Have similar subunit structures and enzyme requirements.
 Possess cutting and methyltransferase functions.
 Example: BseMII from Bacillus stearothermophilus.
-ADVANCE CONCEPT
Type IIS Restriction Endonucleases (Shifted Cleavage):
 Recognize 5- to 7-bp nonpalindromic sequences.
 Cut DNA within 20 bases of the recognition site.
 Examples: Fok I, Alw1.
Type IIT Restriction Enzymes:
 Composed of two subunits, each containing one catalytic
site.
Type IIM Restriction Enzymes
 Examples: Dpn1.
 Cut methylated DNA.
 Useful tool in detecting methylation in DNA.
ADVANTAGES OF BLUNT ENDS
 Blunt ends formed by different enzymes can be joined
regardless of the recognition site.
 Suitable for in vitro recombination with different DNA
fragments.
CONVERSION OF STICKY ENDS TO BLUST NDS
 Sticky ends can be converted to blunt ends using DNA
polymerase to extend the recessed strand in a sticky end,
using the nucleotides of the overhang as a template.
 Alternatively, a single-strand exonuclease can be used to
remove the overhanging nucleotides.
USE OF SYNTHETIC SHORT DNA FRAGMENTS
(ADAPTORS)
 Synthetic short DNA fragments with one blunt end and
one sticky end (adaptors) can be used to convert blunt
ends to specific sticky ends.
APPLICATIONS OF RESTRICTION ENZYMES
 Used for mapping DNA fragments.
 The collection of fragments generated by digestion of a
given DNA fragment with several restriction enzymes is
unique to that DNA.
 Basis for forensic identification and paternity testing using
restriction-fragment analysis of human DNA.
-ADVANCE CONCEPT
CRISPR-Cas Protein-Based Immune Systems:
 Function in about half of all bacteria and 90% of archaea.
 Utilize clustered, regularly interspaced, short palindromic
repeats (CRISPR) and Cas proteins.
 Recognize exogenous DNA and maintain sequence
records of previous infections.
CRISPR Structure:
 Mediated by DNA sequence arrays flanked by Cas genes.
 Cas genes encode nucleases and helicases.
Types of CRISPR-Cas Structures:
 Class 1 (Types I, III, and IV): Have multiple subunit
complexes.
 Class 2 (Types II, V, and VI): Have single subunits.
Integration of Foreign DNA:
 Foreign DNA is inserted into the host's genome at
sequence arrays or spacers.
 Sequences matching host spacers are proto-spacers.
 Proto-spacers are recognized by the host through a 2-5
nucleotide protospacer adjacent motif (PAM) next to the
protospacer sequence.
DNA LIGASE
Function of DNA Ligase:
 Catalyzes the formation of a phosphodiester bond
between adjacent 3′-hydroxyl and 5′-phosphoryl
nucleotide ends.
 Essential for sealing breaks in the DNA backbone during
replication, recombination, and repair processes.
Discovery:
 Predicted by the observation of replication, recombination,
and repair activities in vivo.
 Discovered independently in five different laboratories in
1967.
Enzyme Activity:
 Catalyzes the end-to-end joining of separated strands of
DNA.
RECOMBINATION IN SEXUALLY REPRODUCING
ORGANISMS
 Recombination in Genetics
 Recombination involves the mixing and assembly of
new genetic combinations through crossing over or
physical exchange between molecules.
 A recombinant molecule or organism possesses a
novel combination of DNA sequences.
 Natural Recombination in Sexually Reproducing
Organisms:
 In sexually reproducing organisms, each generation
represents a new combination of parental genomes,
enhancing genetic diversity.
 Recombination occurs during meiosis through
crossing over or breakage and reunion of DNA
duplexes, leading to newly recombined
chromosomes.
 Recombined chromosomes are randomly assorted
into gametes, resulting in offspring with a unique
combination of parental genes.
 Recombinant DNA Technology:
 Recombinant DNA technology involves controlled
mixing of genes, allowing for the alteration,
replacement, deletion, or movement of single genes
into new genomes.
 This directed diversity enables the creation of
organisms with predictable traits, akin to natural
purebreds but with single-gene differences.
 Recombinant DNA technology has implications in
various fields, including laboratory research and
potential applications in disease treatment and
prevention, such as gene therapy.
RECOMBINATION IN ASEXUAL REPRODUCING
ORGANISMS
 Gene Movement in Laboratory Studies:
 Laboratory manipulation of genes originated from
the investigation of natural recombination in
asexually reproducing bacteria.
 In asexually reproducing organisms, genetic
information can be recombined through three
mechanisms: conjugation, transduction, and
transformation.
CONJUGATION
 Conjugation in Bacteria:
 Bacteria involved in conjugation are categorized into
two types or sexes: F+ and F−.
 Conjugation requires physical contact between F+
and F− cells, as demonstrated by experiments
where separation prevents mating.
 Microscopic observation reveals a filamentous
bridge between mating bacteria.
 Research by J. Lederberg and William Hayes
showed that genetic information can move from F+
to F− bacteria, but not vice versa.
 F+ bacteria possess a "fertility factor" responsible for
transferring genetic information and establishing
physical connections during mating.
 After mating, F− bacteria acquire the fertility factor,
becoming F+.
 F Factor in Conjugation:
 The F factor is an extrachromosomal circle of
double-stranded DNA or plasmid carrying genes for
building the mating bridge.
 Genes on the F factor are transferred across the
bridge and replicated, leaving one copy in the F+
bacteria and sending the other into the F− bacteria.
 After mating, both bacteria become F+.
 The F factor may be lost or cured during cell division,
converting F+ bacteria to F−.
 Insertion into Host Chromosome:
 The F factor can insert into the host chromosome
through crossover or recombination.
 As part of the chromosome, it maintains its ability to
direct mating.
 It can carry part or all of the host chromosome
across the bridge into F− bacteria.
 -High-Frequency Recombination (Hfr) Bacteria:
 Strains with chromosomally embedded F factors are
called Hfr bacteria.
 These strains rarely occur but can pull host
chromosomal information into recipient bacteria
during mating.
 Recombination events can insert this information
into the recipient chromosome, forming new gene
combinations.
 Hfr bacteria were used in the first mapping studies.