Packaging of DNA into 

chromosome
• A somatic human cell has 46 chromosomes with a total length of DNA equal to 2 m
but the size of the nucleus is millions of times smaller. 
• Such an enormous amount of DNA must be packaged by an elaborate procedure into
the nucleus and then in chromosomes of the dividing nuclei with precision.
• DNA is associated with a group of basic proteins called histones to form a nuclear
protein complex having about 50% DNA and 50% protein. 
• It is called chromatin. Chromatin stains with basic dyes ( example- Haematoxylin ,
Fuschin )
• It occurs in two forms in the resulting nuclei ;-
• Euchromatin - with a loose structure and is lightly stained
• B.  Heterochromatin- more condensed and deeply stained
• Only Euchromatin is generally active but in dividing since the chromatin condenses
and assembles into specific number of well-defined chromosome for the proper
distribution of replicated DNA among the daughter nuclei or cells
• HISTONES 
• There are five types of histones associated with DNA in the ratio of 1H1 : 2H2a : 2H3
: 2H4 .
• They are rich in arginine and histidine that provide them a positive charge which
forms ionic bonds with negative charges on the phosphate groups of DNA.
• The amino acid sequences of eukaryotic histones are highly conserved in different
groups with little variations.

• However H1 histones shows variation in structure in different species and even in

different tissues of the same organism

• NUCLEOSOMES
• Two molecules each of H2 A, H2B, H3 and H4 histones form an Octamer complex
called a nucleosome core around which the DNA wraps 2 turns in a left-handed
orientation. 
•  DNA enters and leaves the nucleosomes at the same vicinity where one molecule of
H1 histone may seal it virtually all of DNA is associated with nucleosomes. 
• During the interphase the chromatin appears as Beads on string structure with
nucleosome is connected by an intervening stretch of DNA of a variable length of 50–
60 base pairs. 
• It is called linker DNA and is associated with the H1 histone.
• DNA forms 1.65 turns around each nucleosome which is called core DNA and is
resistant to digestion by the nuclease
 • Hierarchy of packaging of DNA
• There are several levels of organisation that result in tight packaging of DNA into the
chromosomes
• A) Wrapping around the nucleosomes :- In the first step extended LinkedIn and
makes to complete turns around each nucleosome ( = 200 bp ) and is sealed by H1
molecule. It results in seven fold compaction of DNA. The nucleosomes  come close
together to form a 10 nm  thick fiber
• B) Formation of 30 nm thick fibre or Chromosome :-  The 10 nm fibre is condensed
further like a spring to form a 30 nm fiber. There are six nucleosomes  per turn which
are organised into a solenoid. Histone H1 is required for the formation of the 30 nm
fiber. The packaging ratio of DNA at this level is about 40 fold
• C ) Formation of Metaphase chromosome from 30 nm fibre :-
• The 30 nm fibre or chromonema forms loops on the central non-histone protein
scaffold of the chromosome. 
• Average length of loops in the loop domain is about 50,000 to 1,00,000 bp of DNA.
• The packaging ratio of the local domain is about 750 fold which is of typical
Euchromatin. 
• In heterochromatin and dividing chromosomes the looped  domains are further
condensed to give it still higher packing ratio. 
• At metaphase a typical human chromosome measures about 4 to 5 micro metres in
length containing about 75nm  of DNA when fully extended. The packing ratio falls
in the range  of 15,000 to 20,000

DNA replication in prokaryotes

DNA replication:-

DNA replication is fundamental process occurring in all living organism to copy their DNA. 
The process is called replication in sense that each strand of ds DNA serve as template
for reproduction of complementary strand

General feature of DNA replication

• DNA replication is semi conservative
• It is bi-directional process
• It proceed from a specific point called origin
• It proceed in 5’-3’ direction
• It occur with high degree of fidelity
• It is a multi-enzymatic process

DNA replication occurs by three steps :-

1. Initiation:
• Initiation complex formation
• Closed complex formation
• Open complex formation
2. Elongation:
• Leading strand synthesis
• Lagging strand synthesis
3. Termination

Initiation:

 DNA replication begins from origin. 

 In E coli, replication origin is called Ori-C which consists of 245 base pair and
contains DNA sequences that are highly conserved among bacterial replication
origin. 
Two types of conserved sequences are found at Ori-C, three repeats of 13 bp
(GATRCTNTTNTTTT) and four/five repeats of 9 bp (TTATCCACA) called 13 mer and 9
mer respectively
 About 20 molecules of Dna A proteins binds with 9 mer repeats along with ATP which
causes DNA to wraps around dnaA protein forming initial complex. The dna A protein
and ATP trigger the opening of 13 mer repeats froming open complex.
 Two copies of dnaB proteins (helicase) binds to 13 mer repeats. This binding is
facilitated by another molecule called dnaC. The dnaB-dnaC interaction causes dnaB
ring to open which binds with each of the DNA strand. The hydrolysis of bound ATP
release dnaC leaving the dnaB bound to the DNA strand.
The binding of helicase is key step in replication initiation. dnaB migrates along the single
stranded DNA in 5’-3’ direction causing unwinding of the DNA
 The activity of helicase causes the topological stress to the un winded strand
forming supercoiled DNA. This stress is relieved by the DNA topoisomerase (DNA
gyrase) by negative supercoiling. Similarly, single stranded binding protein binds
to th separated strand and prevents reannaeling of separated strand and stabilize the
strand.
 The DNA polymerase cannot initiate DNA replication. So, at first primase synthesize
10±1 nucleotide (RNA in nature) along the 5’-3’ direction. In case of E.coli primer
synthesized by primase starts with ppp-AG-nucleotide. Primer is closely associated
with dnaB helicase so that it is positioned to make RNA primer as ssDNA of lagging
strand
 Elongation:
1. Leading strand synthesis

 Leading strand synthesis is more a straight  forward process which begins with the
synthesis of RNA primer by primase at replication origin.
 DNA polymerase III then adds the nucleotides at 3’end.
 The leading strand synthesis then proceed continuously keeping pace with unwinding
of replication fork until it encounter the termination sequences.

Lagging strand synthesis:
 The lagging strand synthesized in short fragments called Okazaki fragments. 
 At first RNA primer is synthesized by primase and as in leading strand DNA
polymerase III binds to RNA primer and adds dNTPS.
On this level the synthesis of each okazaki fragments seems straight forward but the reality is
quite complex

Mechanism of Lagging strand synthesis :- 

 The complexicity lies in the co-ordination of leading and lagging strand synthesis.
  Both the strand are synthesized by a single DNA polymerase III dimer which
accomplished the looping of template DNA of lagging strand synthesizing Okazaki
fragments.
 Helicase (dnaB) and primase (dnaG) constitute a functional unit within replication
complex called primosome.
DNA pol III use one set of core sub unit (Core polymerase) to synthesize leading strand and
other set of core sub unit to synthesize lagging strand
 In elongation steps, helicase in front of primase and pol III, unwind the DNA at the
replication fork and travel along lagging strand template along 5’-3’ direction.
 DnaG primase occasionally associated with dnaB helicase synthesizes short RNA
primer. A new B-sliding clamp is then positioned at the primer by B-clamp loading
complex of DNA pol III.
 When the Okazaki fragments synthesis is completed, the replication halted and the
core sub unit dissociates from their sliding clamps and associates with new clamp.
This initiates the synthesis of new Okazaki fragments.
 Both leading and lagging strand are synthesized co-ordinately and simultaneously by
a complex protein moving in 5’-3’ direction. In this way both leading and lagging
strand can be replicated at same time by a complex protein that move in same
direction.
 Every so often the lagging strands must dissociates from the replicosome and
reposition itself so that replication can continue.
 Lagging strand synthesis is not completes until the RNA primer has been removed
and the gap between adjacent Okazaki fragments are sealed. The RNA primer are
removed by exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA. The gap
is then sealed by DNA ligase using NAD as co-factor.

Termination

 Eventually the two  replication fork of circular E. coli chromosome meet at

termination recognizing sequences (ter).
The Ter sequence of 23 bp are arranged on the chromosome to create trap that the replication
fork can enter but cannot leave. Ter sequences function as binding site for TUS protein
  Ter-TUS complex can arrest the replication fork from only one direction. Ter-TUS
complex encounter first with either of the replication fork and halt it. The other
opposing replication fork halted when it collide with the first one. This seems the Ter-
TUS sequences is not essential for termination but it may prevents over replication by
one fork if other is delayed or halted by a damage or some obstacle.
 When either of the fork encounter Ter-TUS complex, replication halted.
 Final few hundred bases of DNA between these large protein complexes are replicated
by not yet known mechanism forming two interlinked (cataneted) chromosome.
 In E. coli DNA topoisomerase IV (type II) cut the two strand of one circular DNA and
segrate each of the circular DNA and finally join the strand.
The DNA finally transfer to two daughter cell

DNA replication in eukaryotes

DNA replication is the process of producing two identical replicas from one original DNA
molecule.

Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to only
once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the
duplication of a cell and is necessary for the maintenance of the eukaryotic genome. DNA
replication is the action of DNA polymerases synthesizing a DNA strand complementary to
the original template strand. To synthesize DNA, the double-stranded DNA is unwound by
DNA helicases ahead of polymerases, forming a replication fork containing two single-
stranded templates. In G1 phase of the cell cycle, many of the DNA replication regulatory
processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S
phase of the cell cycle, and the entire genome must be unwound and duplicated to form two
daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally,
one copy of the genomes is segregated to each daughter cell at mitosis or M phase. These
daughter copies each contain one strand from the parental duplex DNA and one nascent
antiparallel strand.

This mechanism is conserved from prokaryotes to eukaryotes and is known as

semiconservative DNA replication. The process of semiconservative replication for the site of
DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is
open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for
the incorporation of free nucleotides into double-stranded DNA.
Three major steps in eukaryotic DNA replication
I. Initiation
Initiation of eukaryotic DNA replication is the first stage of DNA synthesis where the DNA
double helix is unwound and an initial priming event by DNA polymerase α occurs on the
leading strand. The priming event on the lagging strand establishes a replication fork.
Priming of the DNA helix consists of synthesis of an RNA primer to allow DNA synthesis by
DNA polymerase α. Priming occurs once at the origin on the leading strand and at the start of
each Okazaki fragment on the lagging strand.
 Pre-replicative Complex

Origin Recognition Complex

The first step in the assembly of the pre-replication complex (pre-RC) is the binding of the
origin recognition complex (ORC) to the replication origin.
Cdc6 Protein
Binding of the cell division cycle 6 (Cdc6) protein to the origin recognition complex (ORC)
is an essential step in the assembly of the pre-replication complex (pre-RC) at the origins of
replication. Cdc6 binds to the ORC on DNA in an ATP-dependent manner, which induces a
change in the pattern of origin binding that requires Orc10 ATPase. Cdc6 requires ORC in
order to associate with chromatin and is in turn required for the minichromosome
maintenance proteins (Mcm2-7) to bind to the chromatin. The ORC-Cdc6 complex forms a
ring-shaped structure and is analogous to other ATP-dependent protein machines. The levels
and activity of Cdc6 regulate the frequency with which the origins of replication are utilized
during the cell cycle.
Cdt1 Protein
In fission yeast and Xenopus, chromatin licensing and DNA replication factor 1 (Cdt1)
protein is required for the licensing of chromatin for DNA replication. Cdt1 is essential for
DNA replication and performs its role during the formation of the pre-replicative complex by
loading the minichromosome maintenance proteins (Mcm) into the chromosome.
Minichromosome Maintenance Protein Complex
The assembly of the minichromosome maintenance (Mcm) proteins function together as a
complex in the cell. The assembly of the Mcm proteins onto chromatin requires the
coordinated function of the Origin Recognition Complex (ORC), Cdc6, and Cdt1. Once the
Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the
chromatin without preventing subsequent DNA replication. This suggests that the primary
role of the prereplication complex is to correctly load the Mcm proteins. The Mcm proteins
support roles both in the initiation and elongation steps of DNA synthesis.
Initiation Complex
During the G1 stage of the cell cycle, the replication initiation factors, origin recognition
complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex,
bind sequentially to DNA to form the pre-replication complex (pre-RC).
DDK and CDK Kinases
At the onset of S phase, the pre-replicative complex must be activated by two S phase-
specific kinases in order to form an initiation complex at an origin of replication. One kinase
is the Cdc7- Dbf4 kinase called Dbf4-dependent kinase (DDK) and the other is cyclin-
dependent kinase (CDK).

II. Elongation
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3' end
of the newly synthesized polynucleotide strand. The template strand specifies which of the
four DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the
nucleotide complementary to the template nucleotide at that position is added to the new
strand.
Eukaryotic replisome complex and associated proteins.
Replication Fork
The replication fork is the junction the between the newly separated template strands, known
as the leading and lagging strands, and the double stranded DNA

III. Termination
Eukaryotic chromosomes have multiple origins of replication, which initiate replication
almost simultaneously. Each origin of replication forms a bubble of duplicated DNA on
either side of the origin of replication. Eventually, the leading strand of one replication
bubble reaches the lagging strand of another bubble, and the lagging strand will reach the 5'
end of the previous Okazaki fragment in the same bubble.
DNA polymerase halts when it reaches a section of DNA template that has already been
replicated. However, DNA polymerase cannot catalyze the formation of a phosphodiester
bond between the two segments of the new DNA strand, and it drops off. These unattached
sections of the sugar-phosphate backbone in an otherwise full-replicated DNA strand are
called nicks.
Once all the template nucleotides have been replicated, the replication process is not yet
over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate
backbone need to be connected.
Difference between prokaryotic and eukaryotic DNA replication
Prokaryotic DNA Replication Eukaryotic DNA replication
Occurs inside the cytoplasm Occurs inside the nucleus
Only one origin of replication per molecule ofHave many origins of replication in each
DNA chromosome
Origin of replication is about 100-200 or more Each origin of replication is formed of about
nucleotides in length 150 nucleotides
Replication occurs at one point in each Replication occurs at several points
chromosome simultaneously in each chromosome
Only one replication fork is formed Multiple replication forks are formed
simultaneously in each chromosome
Only have one origin of replication Has multiple origins of replication
Initiation is carried out by protein DnaA and Initiation is carried out by the Origin
DnaB Recognition Complex

DNA gyrase is needed DNA gyrase is needed

Replication is very rapid Replication is very slow

Genetic code
s The genetic code is the set of rules used by living cells to translate information encoded
within genetic material  sequences of nucleotide triplets, or codons) into proteins.
Translation is accomplished by the ribosome, which links proteinogenic amino acids in an
order specified by messenger RNA (mRNA), using transfer RNA (tRNA) molecules to carry
amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly
similar among all organisms and can be expressed in a simple table with 64 entries.encoded
as

Codons: 3 base code for the production of a specific amino acid, sequence of three
of
Properties of genetic code
The properties or the characteristics of the genetic code are stated below:

 The genetic code is the set of rules which dictates the linear sequence of nucleotides
in the linear sequence of a polypeptide.
 They specify how a nucleotide sequence of an mRNA is translated into the amino acid
sequence of a polypeptide
 Thus it explains the relationship between nucleotide sequences is of the mRNA and
the amino acid sequence of the polypeptide.
 The genetic code consists of 64 different codons and each code for 1 of the 20 amino
acids.
 Codons can be defined as a group of 3 nucleotides which is read by a cell to decode
an mRNA
a) Most codons specify an amino acid
b) The start codon AUG marks the beginning of a protein
c) The stop codon marks the end of a protein
d) The codons are read during translation, beginning at the start codon till the stop
codon. These mRNA codons are read from 5’ to 3’ and they specify the order of
amino acids in proteins from the N-terminus to the C-terminus.
 Triplet nature: The triplet nature of the genetic code explains that singlet and
doublet codes are not adequate to code for 20 different amino acids.
 Degeneracy of genetic code: 
a) The genetic code is degenerate, this means that same amino acid is coded by more
than one base triplet.
b) It does not imply a lack of specificity in protein synthesis, it just describes that one
amino acid can be directed to its place in the amino acid sequence by more than one
base triplets.
c) The amino acids, arginine, alanine and leucine have 6 same codons.
d) There are two types of degeneracy observed in the genetic code: partial and complete.
e) In partial degeneracy, the first 2 nucleotides are identical by the 3rd nucleotide differs.
Example: CUU and the CUC codon for leucine.
f) Complete degeneracy is observed when any of the bases can take the 3 rd position but
still code for the same amino acid. Example: UCU, UCC, UCG and UCA all code for
serine.
 The non-overlapping nature of genetic code: The genetic code is non-overlapping,
which means, two adjacent codons do not overlap each other
a) A non-overlapping code refers to the same letter not being used for two different
codons. In other terms, a single base cannot take part in the formation of more than
one codon.
 The comma less feature of genetic code: The entire code is comma less and there is
no signal to indicate the beginning of the end of a codon.
Also, there are no intermediary nucleotides between the codons.
 Non-ambiguity: The genetic code is non-ambiguous which means a particular codon
will always code for the same amino acid.
 The same amino acid can be coded by more than one codon but the same codon cannot
code for two or more different amino acids.
 The universality of the genetic code: This means the same sequences of 3 bases
encode the amino acids in all life forms from simple to complex organisms.
i. The entire code is based on a study conducted on E. coli, however, it is valid for
organisms.
ii. Only minor exceptions are yeast, mitochondria and the Mycoplasma.
 Polarity: The genetic code has polarity and the code is always red in a fixed
direction. It is read from 5’ to 3’
If the code is read in the opposite direction (i.e., 3′ → 5′), it would specify 2 different
proteins, since the codon would have reversed base sequence.

 A codon in messenger RNA is either translated into an amino acid or serves as a

tradinitional start/stop signal

Codon : 3 base code for the production of a specific amino acid , sequence of three of the four
different nucleotides.
Since there are 4 bases and 3 positions in each codon. there are 4×4×4=64 possible codons
64 codons but only 20 amino acids, therefore most have more than 1 codon
3 of the 64 codons are used as STOP signals they are found at the end of every gene and mark
the end of the protein.
One codon is used as a START signal: it is at the start of every protein
Universal : in all living organisms