1 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

BRIEF HISTORY
 Gregor Mendel (heredity)
 Thomas Hunt Morgan (flies, linkage)
 Frederick Griffith (1928: transformation and mice)
 Oswald Theodore Avery, Colin MacLeod and Maclyn
McCarty (1944: DNA as the transforming agent)
 Erwin Chargaff (late 40’s-early 50’s: base pairing=AT
CG)
 Alfred Hershey-Martha Chase (1952: DNA is not a
protein)
 Watson and Crick (1953: chemical structure of DNA –
secondary structure: double-helix)
 Meselson-Stahl (mid 1950’s: DNA Replication details:
semi-conservative replication model)
 Friedrich Miescher in 1869
 Isolated what he called nuclein from the nuclei of pus
cells
 Nuclein was shown to have acidic properties, hence
it became called nucleic acid

Chromosomes, Genes, and Nucleic Acids

 Chromosomes
 structure in the cell nucleus which is the visible
carrier of genetic information
 Genes
 portion of a chromosome that controlled a specific
inheritable trait
 Nucleic Acids
 carries information which directs the process of
protein synthesis
 within the nucleic acids are the codes needed for
transcription and translation of proteins
Genome (entire set of genes of an organism) size is based
on number of nucleotide pairs present
 Nucleotides are monomers of nucleic acids
 DNA is made up of repeating series of nucleotides
The presence of cells makes a thing living.
Chromosomes within these cells makes it capable of doing
biological processes. Within these chromosomes are genes
that codes for specific proteins. Those genes are made up of
nucleic acids.
Long strands of nucleic acid (DNA) is organized
through the protein histones. With the help of histones, the
DNA will be wrapped properly to be organize and form
genes. Then, it will be further wrapped and organized to form
a chromosome.
Every specie have 2 sets of chromosomes (pairs)
which are tied together through a protein called centromere.
The end of the chromosome is called a telomere, which are
made up of genes that are not coding for specific proteins
(protective caps of chromosomes because the sequences
present are not functional)

 C-value paradox: Among eukaryotes, there is no

consistent relationship on the C-value (DNA content of
the genome) and the metabolic, developmental, or
behavioural complexity of the organism

Criteria for the Genetic Material

1. It must contain information that controls the synthesis of
enzymes and other proteins;
 Base pairs present within the DNA codes for amino
acids which are needed for the synthesis of proteins
2. It must be able to self-replicate with high fidelity, yet allow
a low level of mutation;
 its base pairs are complementary to each other,
making a complementary pair using a single strand
of DNA alone
 Within the nucleus, chromosomes are located (as pairs)
 replication of DNA allows low level of mutation
allowing more room for improvement  One set from the mother, one set from the father
3. It must be located in the chromosomes, which had been  Chromosomes are tied together (by protein centromere)
found to carry traits from one generation to the next  Ends of the chromosome (Telomeres)
 Chromosomes are made up of DNA, they are also  Within the chromosome are genes (genes are specific
located in the nucleus to protect it from cellular portions of chromosome coding for a protein which
damage functions in various phase)
 In the genes are nucleic acids (DNA: double-helix
molecule containing base pair)
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 DNA are wrapped around histones to be organized

 2 types of histone:
1. Responsible for organizing nucleosome (8 histones
with DNA wrapped around it)
2. Responsible for building up solenoid (structure made
up of 6 nucleosome and 6 histones)

Composition of Nucleic Acids

 Nucleic Acids (repeating series of nucleotide)
 Polymers (polynucleotides)

 Parts of Nucleotide
 A five-membered ring monosaccharide
 A nitrogen-containing cyclic compound (nitrogenous
bases)
 A phosphate group

 Types of Nucleic Acids

 DNA (genetic material – doesn’t function without
RNA)
 RNA
 Sugar + Base = Nucleoside
 Adenine + (deoxy)ribose = (deoxy)adenosine
 Guanine + (deoxy)ribose = (deoxy)guanosine
 Cytosine + (deoxy)ribose = (deoxy)cytidine
 Thymine + deoxyribose = deoxythymidine
 Uracil + ribose = uridine

 Sugar + Base = NUCLEOSIDE

 Sugars
 2-deoxyribose (for DNA)
o 5th carbon – phosphate group
o 3rd – next nucleotide attached
 Ribose (for RNA)
o 2nd carbon - oxygen
 Nitrogenous Base
 Purines (2)
o Contains two-fused N-containing ring
 Adenine (A) o Adenosine: nucleoside formed after combination of
adenine with ribose
 Guanine (G)
 Pyrimidines (3)
o Has one nitrogen-containing ring
 Cytosine (C)
 Thymine (T)
 Uracil (U)
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Bases Ribonucleosides Ribonucleotides

Adenine (A) Adenosine Adenosine 5’-
Monophosphate
(AMP)
Guanine (G) Guanosine Guanosine 5’-
Monophosphate
(GMP)
Cytosine (C) Cytidne Cytidine 5’-
Monophosphate
(CMP)
Uracil (U) Uridine Uridine 5’-
Monophosphate
(UMP)

 NUCLEOSIDE + Phosphate = NUCLEOTIDE Structures of Nucleic Acids

 Are the building blocks of nucleic acids
 Monomers of the DNA and RNA polymers
 Is a 5’-monophoshpate ester of a nucleoside
 Are named by adding 5’-monophosphate at the end
of the name of the nucleoside
 Nucleotides
 Can add additional phosphate groups to form
diphosphate or triphosphate esters (necessary to
produce energy needed in transcription, translation
and repli)
 Primary Structure (from polymerization of monomers)
 the repeating sequence of nucleotides form its
primary structure (forming alternating ribose and
phosphate backbone – providing structural stability)
 Nucleotides are connected through Carbon-3 of
ribose sugar (repeated until nucleic acid is form)
 Alternating deoxyribose and phosphate group
 Backbone of the molecule
 Provides structural stability
 the bases that are the side-chain groups
 carry all the information necessary for
protein synthesis
 Secondary Structure
 Based on:
o Chargaff rule
 A, T, G, and C (complimentary) are present
in equimolar quantities (refers to similarity in
molar concentration in DNA hydrolysis;
equal concentration)
 If this 2 molecules are placed together, they
form hydrogen bonds
 Similar molar concentration upon DNA
hydrolysis
o X-ray diffraction photographs
 Obtained by Rosalind Franklin and Maurice
Wilkins
 Diagonal image: helical structure of DNA

Bases Deoxyribonucleosides Deoxyribonucleotides

Adenine Deoxyadenosine Deoxyadenosine 5’-
(A) Monophosphate
(dAMP)
Guanine Deoxyguanosine Deoxyguanosine 5’-
(G) Monophosphate
(dGMP)
Cytosine Deoxycytidine Deoxycytidine 5’-
(C) Monophosphate  Double helix
(dCMP) o The 2 (single strand of DNA) polynucleotide
chains run in opposite directions
Thymine Deoxythymidine Deoxythymidine 5’-
o One 5’ – OH and one 3’ – OH terminal
(T) Monophosphate
o Bases are hydrophobic (non polar, tucked
(dTMP)
inside)
o Sugar phosphate backbone is hydrophilic (polar,
exposed to environment)
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o Sugar-phosphate backbone is exposed to the

aqueous environment

 Higher Structure 3 forms of the helical structure of DNA: A, B, Z

 Histones  X-ray photograph (Franklin and Wilkins) of DNA was in
o Basic protein to w/c the DNA is coiled around B form (common structure of DNA, followed by A)
 Nucleosome  Z form is rarest and is only obtained in experiments
o Further arrangement of DNA in order to organize
them in the chromosomes
o 8 histones wrapped together by DNA
 Histones are attracted to the DNA through
electrostatic force (basic protein = (+)
charge; DNA is acidic = (-) charge)
o Acidic DNA + basic histones
o Attract each other by electrostatic (ionic) forces

Hydrogen bonds present in base pairs:

 A form – 11 base pairs before helix rotates

 B form – 10 base pairs before helix rotates
 in both A&B, glycosidic bonds are in anti-manner or
anti-orientation;
o syn-orientation (glycosidic bond is above the
ribose)
o anti-orientation (nitrogenous base below the
 3 hydrogen bonds ribose)
 Z form – 12 base pairs before helix rotates (glycosidic
bonds are anti and syn)

Ribonucleic Acids
 Single strand nucleic acids
 Usually located outside the nucleus (but made inside the
nucleus and transported to cytoplasm to do its function)
 The important intermediary player in the central dogma
 The only genetic material of viruses (viruses are
classified as non-living things because it only has one
genetic material)
 2 hydrogen bonds (easier to breakdown
than G and C pair)
Types of Ribonucleic Acids
 Messenger RNA (mRNA)
 Codes for protein
 Ribosomal RNA (rRNA)
 Forms the core of the ribosomes
 5 CYTOGENETICS
 Machinery for making proteins
 Transfer RNA (tRNA)
 Matches code for amino acid on mRNA and position
the right amino acid in place during protein synthesis
 Transfers free amino acids to the polypeptide chain
 Ribozyme
 RNA with enzymatic properties
 Functions in mRNA splicing
mRNA carries the code needed for the production of
specific protein, once it goes into the cytoplasm it merges
with rRNA to be read. The tRNA will carry the corresponding
amino acid coded by the codon present on the mRNA. Codon
are the 3 consecutive nitrogenous base present in the
mRNA.
Each codon codes for a specific amino acids. The
delivery of the tRNA of this specific amino acid to the rRNA
will now initiate the formation of primary structure of protein.
It is only the mRNA that can traverse the nucleus and
cytoplasm. Since, tRNA and rRNA is only present in the
cytoplasm. Before mRNA is transferred to the cytoplasm it
undergoes a series of changes for it to be more refined and
efficient in being the code for protein formation.
 Messenger RNA (mRNA)
 Carry the genetic information from the DNA in the
nucleus directly to the cytoplasm
 Consists of a chain of nucleotides whose sequence
is exactly complementary to one of the strands DNA
NUCLEIC ACID & REPLICATION 02/01/2021
Complementary: mRNA is the exact
opposite of that one strand present in the
DNA
 Opposite in the sense of its complementary
base pairs
 If a single strand in the DNA codes for
AAGG, mRNA will code for UUCC
 mRNA now carries the code for the
formation of some protein
mRNA contains untranslated regions (regions that
do not code for amino acids), sequence usually starts few
amino acids from the 5’ end of the mRNA
 Transfer RNA (tRNA)
 Contains 73 to 93 nucleotides per chain
 Can carry a single type of amino acid
 Every amino acid have one tRNA carrier
 There is at least one different tRNA for each of the
20 amino acids
 Transports amino acids to the site of protein
synthesis in the ribosomes
 Most important parts: anticodon arm and acceptor arm
 Anticodon arm – complementary base pairs of mRNA
 3 nitrogenous base pairs present at the end
of tRNA is complementary to the codon
present in mRNA
 Each anticodon corresponds to a specific
type of amino acid carried by the tRNA
 Amino acids are connected to the tRNA at the acceptor
arm (3’ end of tRNA)
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 In the 3rd carbon, the amino acid will attach

to the tRNA

s = Svedberg unit (unit used to determine the sedimentation

rate of the different molecules or compound)
 60s ribosomes (large ribosomes) – it is heavier does
having a faste sedimentation rate

 Antiocodon: CGG
 Complementary to the codon present on the mRNA
(GCC)
 Corresponds to the amino acid alanine
 Amino acid is connected to the 3’ end of transfer RNA
 Structural formula of tRNA:
 Yellow – nitrogenous
bases
 Blue – sugar
phosphate backbone
 Ribozymes
 RNA with enzymatic capabilities
 Catalytic RNA – they can catalyse different reactions
or initiate different reactions specifically splicing of
mRNA
 Catalyse the splicing of mRNA – refers to the
process of removing unnecessary parts of the
mRNA for it to become more efficient in protein
synthesis
How does the Ribozyme do its job?

 Ribosomal RNA (rRNA)

 RNA that is complexed with proteins in ribosomes
 Main component of ribosome
 Complex machinery that is the site of protein
synthesis
 2 subunits
o 1 large – catalyzes the peptide bond formation
o 1 small – binds mRNA and tRNA

 Joining up with mRNA

 It detects or scan which are the specific parts in that
mRNA that should be removed
 Once the ribozyme finds that specific part, it will
catalyse a reaction that will cause the mRNA to
Normally, when a ribosome doesn’t perform protein break at that specific portion that is not necessary for
synthesis, these subunits are separated in the cytoplasm. the coding a specific protein
They will merge once they decided to perform protein
synthesis.
 Small subunit – where mRNA will bind
 Large subunit – amino acids will combine in order to
form primary structure of proteins
 7 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

 The proteins made are needed for various biological

processes and survival

 however, there are instances that ribozymes acts as

regulator in order to prevent the overproduction of  One gene = one protein
proteins  DNA should have the capacity to replicate itself to
 It does that by cutting large portions of the mRNA pass it from a single cell to daughter cells.
 If lots of parts of the mRNA were cut, including the
parts needed for the synthesis of protein, then that
mRNA will not be functional for protein synthesis
- Decreased rate for protein synthesis if ribozyme
cut large parts of the mRNA molecule

Exons and Introns

 Exons
 Coding sequence
 Expressed sequence
 Portion in the mRNA that codes for a specific amino
acid to make protein
 Introns
 Replication or the formation of duplicates of DNA to be
 Noncoding sequence
passed down from the mother to daughter cell is carried
 Part in the RNA that has no purpose; do not code for by DNA Polymerases
proteins  Transcription - process of transcribing DNA template to
 Intervening sequence mRNA is carried out by RNA polymerase
 Where DNA analysis happens  Translation = making proteins from the mRNA template
- Identifies specific identity in a person using DNA is carried out by the ribosomes
(to single out an individual they use introns)  RNA to DNA – enzyme reverse transcriptase (makes
- Because every individual have their own unique DNA from RNA
sequence of introns
 Transcription
 Occurs in the nucleus (to protect mRNA)
 Information encoded in a DNA molecule is copied
into an mRNA molecule
 Translation
 Information encoded in an mRNA molecule is used
to assemble a specific protein

Expression of Genes
 Some genes are transcribed in large quantities because
In splicing, the introns are removed
we need large amount of this protein
- In transcription, the first product is pre mRNA
 Some genes are transcribed in small quantities because
which contains introns
we need only a small amount of this protein
- If these introns is involved in protein translation,
 What makes expression of genes possible is the
protein synthesis won’t be efficient
presence of transcription factors and other proteins
that act intranuclearly (inside the nucleus)
Central Dogma of Life
- Ex. Steroid hormones (activate genes to create
 DNA acts as a “manager” in the process of making
protein), Vit E (retinoic acid binds with retinoid x
proteins
receptor will activate genes to increase
 DNA is the template or starting sequence that is copied
transcription of those genes and protein
into RNA that is then used to make the protein
synthesis will increase)
 8 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

 Cell Cycle
Some genes are overly expressed in some individuals, there - G1 Phase – cell is metabolically active
are also underexpressed in some individuals - S Phase – DNA Replication (8 hours for normal
- This causes the different physical appearance of somatic cells: body cells)
individuals within a specie. - G2 Phase – cell growth continues
- Mitotic phase – where the cell will divide
DNA Replication
 Means to produce molecules that have the same base
seuquence
 To distribute the DNA of the parent cells to its daughter
calls (cells die eventually – It needs to be passed to code
proteins)
 Process that ensures the stability of an organism
 Producing two identical replicas of DNA

 Runs in opposing direction

- For the hydrogen bond between
complementary bases to meet
 DNA are made up of repeating sequences of
nucleotides
 Nucleotides are made of ribose (DNA: 2-
deoxyribose)
 5th carbon – phosphate is connected
 Next nucleotide is connected at Carbon-3 of the
ribose
 5’ to 3’ direction (exposed carbons)
 All DNA present is replicated (needed to pass to the  DNA strands run in opposite direction,
daughter cells) complementary strand runs in 3’ to 5’ direction
 Unwind DNA from binding with histones (remove  Anti-parallelism of the DNA
wrapping through the process of acetylation – makes the hydrogen bonds
addition of acetyl groups to the histone groups to meet
loosen its binding to DNA)
 DNA replication takes place in the S phase (intended for  The DNA backbone
DNA Replication)  Alternating phosphate-sugar
 S phase during interphase of the cell cycle backbone
(interphase and mitotic phase) o Refer to the 3’ and 5’ ends of
 Nucleus of eukaryotes (membrane-bound to protect the DNA
DNA)
 9 CYTOGENETICS
 Anti-parallel strands
 Nucleotides in DNA backbone are bonded from
phosphate to sugar between 3’ and 5’ carbons
o DNA molecules has “direction”
o Complementary strand runs in opposite
direction
 Base pairing
 Base are paired because of hydrogen bonds
 Adenine and Thymine: 2 H bonds
 Guanine and Cytosine: 3 H bonds
DNA Replication Models
 Semiconservative Replication
 DNA Replication would create two molecules
 Each of them would be a complex of an old
(parental) and a daughter strand
 Newly formed molecules is composed of 1 strand
from parent and 1 strand from daughter.
 Conservative Replication
 DNA Replication process would create a brand new
DNA double helix made of two daughter strands
while the parental chains would stay together
 Dispersive Replication
 Replication process would create two DNA double-
chains, each of them with parts of both parent and
daughter molecules
NUCLEIC ACID & REPLICATION 02/01/2021
DNA Replication is semi-conservative
 Replication of DNA
 New strand is half-parent template and half-new
DNA
 Replication is semi-conservative
 This is proven in the experiments of
o Meselsohn and Stahl
o Taylor
Meselsohn and Stahl Experiment
 Grew a bacteria in an isotope (variants of elements with
different molecular weight)
 2 isotopes used:
 N15 – nitrogen weighing 15 amu
 N14 – nitrogen weighing 14 amu
 1st step: grew a bacteria in N15
 Sample of a bacteria from the medium at 0 minutes
 Sample subjected to DNA extraction
 DNA subjected to ultracentrifugation
 DNA settled at the bottom (heavy because it
contains N15)
 They took a sample from N15 and transferred it to N14
 N14 (Light medium)
 After 20 mins (1 cell division cycle), they extracted a
sample from the medium, subjected to DNA
extraction then ultracentrifugation
 It was found out that the DNA in N14 settled at the
middle
 DNA formed is lighter compared to N15
 Labeled it as Intermediate DNA
 After 40 minutes (2 rounds of cell division), another
sample from N14 subjected to ultracentrifugation
 Found out that there are 2 types of DNA formed
- Intermediate DNA (N14 and N15)
- Light DNA (pure n14)
 Result:
 1st test tub is the parent DNA (n15)
 2nd test tube is the first generation of cells from the
parent DNA (newly synthesize DNA)
 3rd test tube: product of 2 replication cycles
 10 CYTOGENETICS
Taylor Experiment
 Utilized a plant species: Vicia faba
- Isolate DNA from that plant
- Adds label (Label – radioactive or fluorescent
attached to a substance you want to test)
- For the experiment, he made a label that will
attach to thymine (H-Thymidine)
- After one round of cell division, one strand of the
chromosome is labelled (parent), and one is not
labelled (daughter dna)
DNA REPLICATION
 As the 2 DNA strands open at the origin, replication
bubbles form
o Prokaryotes (bacteria) have a single bubble
- Only one replication origin is
needed that is because the
chromosomes of prokaryotes are
simple
- Because prokaryotes have single
replication origin. They can form a
single replication bubble (the bubble
formed in replication because of the
separation of DNA strands)
- Any activity requires spaces. In
replication, there is a space formed
in the DNA template because in that
space that the replication happens
and because of that space there is
a formation of replication bubbles
o Eukaryotic chromosomes have many
bubbles
- Multiple replication origins are
needed because the chromosomes
of eukaryotes are way more
complex than prokaryotes
- Have multiple bubbles, because
multiple replication origins they
have multiple replication bubbles.
Because replication happens at
multiple sites in eukaryotic
chromosomes.
NUCLEIC ACID & REPLICATION 02/01/2021
Differentiating replication (prokaryotic vs eukaryotic cells)
 Location
o Eukaryotic
- Replication happens within the
nucleus because the DNA is
located in the nucleus and to
protect the DNA from cellular
damages
o Prokaryotic
- Replication happens within the
cytoplasm. In that specific area of
cytoplasm termed as nucleoid
 Stage of Cell Division
o Eukaryotic
- Replication happens in S Phase in
cell cycle
o Prokaryotic
- Happens prior to binary fusion. So
unlike eukaryotic cells there’s no
specific cell cycle and they do
replication before binary fusion/ cell
division
 Origin
o Eukaryote
- The replication origin are multiple
because of the fact that
chromosomes of eukaryotes are
complicated
o Prokaryotic
- The replication origin is only at a
single point because of the
chromosomes of prokaryotic cells
are simple.
 Replication Bubble
o Eukaryotic
- Numerous due to multiple
replication origin
o Prokaryotic
- One due to single replication origin
 Replication fork
o Eukaryotic
- Numerous due to numerous
replication bubble
o Prokaryotic
- Two due to single replication origin
and single replication bubble
o What is Replication fork?
 11 CYTOGENETICS
- Areas within the replication bubble
wherein the DNA is being
separated in two strands real-time.
- Basically, replication fork is the end
of the replication bubble
 Okazaki Fragments
o Eukaryotic
- Shorter but produced in a slower
rate
o Prokaryotic
- Longer but ironically in faster rate
 Termination Site
o Eukaryotic
- Multiple termination site along the
chromosome corresponding to
each origin of replication
o What is termination
- Part of the replication cycle where
active replication stops
o Prokaryotic
- Single termination site 180 degree
from the origin.
Specific steps in replication cycle (Eukaryotic vs
Prokaryotic)
 Recognition of Origin of Replication
o What is recognition of origin
- In order for replication to begin.
Protein should recognize/mark the
origins of replication
- Origin of Replication is rich with
Adenine, Thymine sequences. That
is because AT sequences contain
2 hydrogen bonds which is easier
to break. Thus, making them the
best site for origin of replication
- AT Sequences site is where DNA
begin its separation
o Eukaryotic
- It is not established what specific
protein is used in marking origin of
replication
o Prokaryotic
NUCLEIC ACID & REPLICATION 02/01/2021
- DNA A Protein
 Unwinding of DNA Double Helix
o Eukaryotic and Prokaryotic
- Helicase (Requires ATP); For both
cells’ helicase will separate
helicase in unwinding the double
helix of DNA
 Stabilization of unwound template strands
o Eukaryotic and Prokaryotic
- Single strand DNA-binding protein;
are used to keep the separated
DNA separated strands and
prevent them from being wounded
again
 Synthesis of RNA primers
o Eukaryotic and Prokaryotic
- Primase
o Eukaryotic
- Its primase is a part DNA
polymerase alpha
 Synthesis of DNA Leading Strand and Lagging
Strand
o Eukaryotic
- Leading strand
 DNA Polymerase δ
- Lagging Strand
 DNA Polymerase α
o Prokaryotic
- Leading strand
 DNA Polymerase III
- Lagging strand
 DNA Polymerase III
 Removal of RNA Primers
o Eukaryotic
- Unknown; Varies from species to
species
o Prokaryotic
- DNA Polymerase I (5’ to 3’
exonuclease);
 Joining of Okazaki Fragments
o Eukaryotic
- DNA Ligase which requires ATP
o Prokaryotic
- DNA Ligase which requires NAD
 Removal of positive supercoils ahead of advancing
replication forks
o Eukaryotic
- DNA Topoisomerase II
o Prokaryotic
- DNA Topoisomerase II (DNA
Gyrase)
Eukaryotic DNA Replication
 Origins of replication is characterized by the
presence of A-T rich sites
o Parts of dna where adenine and thymine
are rich this origins should be marked with
different proteins in order to recruit
 Once the replication proteins are recruited at the
site origin of replication the replication bubbles will
now be formed
 12 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

o Replication bubbles are formed because bacterium to another using various

the DNA strands are already separated mechanism
 The replication bubble will continue to grow until o This plasmid is what makes bacteria
each replication bubble will merge capable of antibiotic resistance
o Replicate through rolling circle replication
 Begins in a protein nicks one strand of a DNA
o That nicking leads to formation of 3 prime
end and 5 prime ends
 A helicase like protein will come to the nick in order
to separate these 2 DNA strands
 A DNA polymerase will bind to 3 prime end in order
to synthesize the new DNA strand.
 So, while helicase is separating the two DNA
strand. This DNA strands will gradually form a circle
 Once it is fully separated it is then furthered
rejoined and from that single DNA strand a news
DNA Strand will form

Prokaryotic DNA Replication

 Begins at a single origin
 From the origin a replication bubble will form
- Within that replication bubble is where
replication happens and the synthesis of
new DNA template happen
 So, the replication bubble will increase in size until
the replication bubble will meet its end in a
termination site
- The site of termination site of prokaryote is  Rolling Circle Replication Beings when RepA goes
180 degree from the site of origin to the site of plasmids wherein the origin will begin
 After the termination of replication there are 2 that specific point in the plasmid is origin of
chromosomes that is formed replication
- Chromosomes are made up of DNA, if the
DNA is replicated so is the chromosomes.
These chromosomes will be distributed in
its daughter cells.

 RepA will create a nick in the DNA strand will for a

3 prime end and a 5 prime end

Rolling Circle Replication

 Prokaryote specifically bacteria contain
extrachromosomal DNA which are called plasmids.
o Plasmid replicate separately from the
bacteria’s main chromosomes
o Plasmids are DNA are way simpler  In the 3 prime end, DNA polymerase III will bind
compared than bacteria’s main
chromosome at the same time these
plasmids can be passed from one
 13 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

- At this point the DNA is double stranded

before a new DNA strand was made

 At the 5-prime end, UVR will bind to separate the

DNA strands.
- While the strand is being separate, single
strand binding proteins will bind to prevent
it from binding or pairing with itself
- While separating, the DNA polymerase III Enzymes in Replication
will catalyze the formation of a new strand.  Helicase
- Unwinds the DNA double helix
- Helicase will cut the paired DNA strands.
Helicase is the equivalent of the UVR in the
Rolling Circle Replication
 Topoisomerase
- Prevents supercoiling; relaxes the part of
the DNA that is not yet separated.
- Types of Topoisomerase
 TYPE I Topoisomerase
 So when it reaches the end of termination, the - Breaks one DNA strand
protein RepA will cut the strand in order to fully and will connect another
separate the two separate strands strand to became a very
loose strand in a part of the
DNA.
- Although the DNA strand is
loose and can be coiled
again it will not be
supercoiled unlike before
 Then a loop will be formed, in order for the DNA - Prevents supercoiling in
polymerase III to begin the synthesis of this new the DNA strand
strand so a 2 copy of plasmid will be formed  TYPE II Topoisomerase
- Breaks double strands of
the DNA and pass another
loop over it
- Prevent supercoiling within
2 DNA pairs

 The DNA ligase will finalize the newly formed DNA

strand in fixing the gaps in order for it to be intact
and continuous

 Primase
- Synthesize short oligonucleotides (primers)
 Clamp Protein (PCNA/Proliferating Cell Nuclear
Antigen for Eukaryote
- A.k.a. Processivity clamps allows the
There are 2 so called origin of the plasmid replication
leading strand to be threaded through.
 Single Stranded Origin
- At the same time makes the process in the
- Present on the separated DNA strand and
leading strand efficient. This clamp protein
while the new DNA is being made for the
helps keep the replication protein in place.
separated DNA strand the DNA template is
 DNA polymerase
single stranded thus making it single
- Joins the assembled nucleotides in order to
stranded
from nucleic acids
 Double Stranded Origin
 14 CYTOGENETICS
- The main enzyme needed in the synthesis
of the new DNA strands
- DNA Polymerase enzymatic activity
 (1) Polymerase
- Polymerizing the new DNA
strand by adding
nucleotides
 (2) Exonuclease
- Break the sugar-phosphate
backbone in the end of a
nucleotide strand
- Proof reading capacity of
DNA polymerase.
- Nucleotides removed from
the ends
 Endonuclease
- Remove nucleotide from
the middle nucleotide
strand
- Internal cuts
 Ligase
- Joins Okazaki fragments in the lagging
strand
Replication
 Initiation
o Formation of Pre-replication complex
formation
- Are the combination of proteins in
the origin site to prepare the origin
site for replication
- ORC (origin recognition complex),
binds to the origin of replication
and that area is AT rich site.
- Once ORC binds to that region the
protein CDC6 and CDT 1 then bind
to it in order to recruit the protein
MCM (minichromosomal
maintenance) helicase-like
o Phosphorylation
- Once MCM binds together with the
proteins it will wait for further
phosphorylation to activate the
complex
- Once it will be added with
phosphate and will activate the
MCM. Thus, the unwinding begins
o Helicase
 Unwinds part and
separates the 2 DNA
strands by breaking the
weak hydrogen bonds
 Remember that
complementary base pairs
are bound by hydrogen
bonds
o Single Strand Binding proteins
 Will now bind to the single
stranded DNA in order to
keep them separated and
to prevent them from
winding back together
NUCLEIC ACID & REPLICATION 02/01/2021
 Attach and keep the 2 DNA
strands separated and
untwisted
o Topoisomerase
 Relieves the tension at the
site where DNA is still
wound in order to relieve
the stress in the DNA
molecule as it separates
 Attaches to the 2 forks of
the bubble to relieve stress
on the DNA molecule as it
separates
 Elongation
o Before new DNA strands can form, there
must be RNA primers present to start the
addition of new nucleotide
- Primers are the starting point of
new DNA strands and are RNA in
nature.
- DNA polymerase need primers
because they cannot add
nucleotides into the new DNA
strand
- RNA primers because if ever there
are errors in the complementary
base pairs it can still be checked
o Primase
- Synthesizes the RNA Primer
o DNA polymerase
- Add the new nucleotides
 As the DNA nucleotides
are synthesized it is still
being checked
- DNA polymerase III
 In prokaryotes the
polymerase that will add
nucleotides is the DNA
polymerase III.
- DNA polymerase a
 While in eukaryotes is DNA
polymerase alpha
 However, synthesis is
complicated in the leading
strand of eukaryotes.
 DNA polymerase Alpha is
changed to DNA
polymerase Delta. Due to
DNA polymerase is not
sticky and has the
tendency to lose focus in
synthesizing the strand.
15 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

That is why processivity

clamps to clump the
different polymerase to the
strand they are
synthesizing
- Replication Factor C (RFC)
assembles Proliferating Cell
nuclear Antigen (PCNA) at the end
of the primer
 PCNA is the processivity o Differentiate the Synthesis of Leading
clamp of the leading Strand (Prokaryote vs Eukaryote)
strand, this protein anchors - Prokaryote
the DNA polymerase Delta  After the primer is added
which is an enzyme that is by the primase. DNA
efficient in building DNA polymerase III will begin
strand than DNA the synthesis of nucleotide
polymerase Alpha - So that DNA polymerase III would
- PCNA displaces DNA pol alpha be effective, clump protein will be
 DNA polymerase alpha will recruited in the site and will clump
be changed into DNA DNA polymerase III to be efficient.
polymerase Delta
- DNA polymerase ẟ binds to PCNA,
recognized and begins leading
strand synthesis
o Build daughter DNA strand
- Add new complementary bases
- DNA polymerase III
- DNA polymerase ẟ

o Eukaryote
- After the primer is added, DNA
polymerase Alpha will add
nucleotides to that primer
- But because DNA polymerase
Alpha is not efficient it will recruit
RCF and PCNA
- PCNA is equivalent of Clump
protein and will displace DNA
polymerase Alpha and will change
it into DNA polymerase Delta which
will be anchored./clump by PCNA
o DNA polymerase can only add nucleotides for DNA pol delta to be efficient
to the 3’ end of DNA - The leading strand has a template
o This causes new strand to be built in a 5’ to running from 5’ to 3’ direction
3’ direction

o The leading strand is synthesized as a

single strand from the point of origin toward
the opening replication fork
- Has a DNA template running in a 3’
to 5’ direction (3 is leading 5)
16 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

o The lagging strand is syn thesized

o Energy for Replication
discontinuously against overall direction fo
- Triphosphate of nucleotides is
replication
where energy for bonding came
o This strand is made in many short
from
segments and is replicated from the
 Nucleotides can be in replication fork toward the origin
triphosphate, from this
nucleotide triphosphate
ENERGY could be obtained
- How? That is when the triphosphate is
broken down. When triphosphate is
separated energy will be released
- So when ATP is converted to AMP
(Adenosine Mono Phosphate) an
energy is released in the process
- Same is true for GTP -> GMP, TTP ->
o Okazaki fragments
TMP, CTP -> CMP
- Series of short segments on the
lagging strand
- Must be join together by an
enzyme ligase

o Ligase joins the okazaki fragment together

to make one strand
o DNA Polymerase can Only add from 3’ to 5’
manner
- Adding bases
 Can only add nucleotides to 3’
end of a growing DNA strand o Proteins (prokaryotes vs Eukaryotes
- Need a starter nucleotide bond
 Strand only grows 5’ -> 3’

 Termination
o The end-replication problem
17 CYTOGENETICS NUCLEIC ACID & REPLICATION 02/01/2021

- Loss of DNA in each eukaryotic

replication cycle because of primer
overhangs
- The answer to this problem are
Telomeres
o Telomeres
- Regions of repetitive DNA close to
the ends and help prevent loss of
genes due to this shortening
- G-C rich

o Telomeric repeat-binding factor

- Binds and stabilizes the double -
stranded telomeric DNA
- Helps the overhangs to form
protective loops
o Telomerase
- Enzyme normally present on stem cells
and germ cells which catalyzes the
formation of telomeres
o Hayflick Limit
- The number of cell division an
organism can make
- Caused by the limited and
consumable presence of telomeres
on somatic cells