DNA replication is the process by which DNA copies itself for cell division. It is semiconservative, meaning each new DNA molecule contains one old and one new strand. Replication occurs through the unwinding of the DNA double helix at an origin of replication by helicase. DNA polymerase then adds complementary nucleotides to each strand, while ligase seals the DNA. Replication of the leading strand is continuous while the lagging strand occurs discontinuously in fragments called Okazaki fragments. Various enzymes and mechanisms ensure replication occurs with high fidelity and accuracy.

2.  DNA carries genetic information from
generation to generation.
 Responsible to preserve the identity of the
species over millions of years.
 DNA may be regarded as Reserve bank of
genetic information or memory bank.

3.  Some viruses contain RNA as the genetic
material
 DNA is more stable than RNA.
 DNA is more suitable molecule for long-term
repository of genetic information.

4.  The biological information flows from DNA to RNA,
& from there to proteins.
 This is central dogma of life.
 DNA in a cell must be duplicated (replicated),
maintained & passed down accurately to the
daughter cells.
DNA RNA Protein
Replication
Transcription Translation

5.  DNA is the genetic material.
 When the cell divides, the daughter cells
receive an identical copy of genetic
information from the parent cell.
 Definition:
 Replication is a process in which DNA copies
itself to produce identical daughter molecules
of DNA with high fidelity.

6.  Replication is semiconservative:
 The parent DNA has two strands
complementary to each other.
 Both the strands undergo simultaneous
replication to produce two daughter
molecules.

7.  Each one of the newly synthesized DNA has
one-half of the parental DNA (one strand
from original) & one half of new DNA.
 This is known as semiconservative
replication - “half of the original DNA is
conserved in the daughter DNA”.
 Experimental evidence was provided by
Meselson & Stahl (1958)

9.  The initiation of DNA synthesis occurs at a
site called origin of replication.
 In prokaryotes, only one site, where as in
eukaryotes, there are multiple sites of origin.
 These sites mostly consist of a short sequence
of A-T base pairs.

11.  A specific protein called dna A(20-50
monomers) binds with the site of origin for
replication.
 This causes the double-stranded DNA to
separate.

12.  Two complementary strands of DNA
separate at the site of replication to form a
bubble.
 Multiple replication bubbles are in
eukaryotic DNA molecules, which is essential
for a rapid replication process.

14.  For the synthesis of new DNA, a short
fragment of RNA (5-50 nucleotides, variable
with species) is required as a primer.
 The enzyme primase (a specific RNA
polymerase) in association with single-
stranded binding proteins (SSBP) forms a
complex called primosome & produces RNA
primers.

15.  A constant synthesis & supply of RNA
primers should occur on the lagging strand
of DNA
 On leading strand only one RNA primer is
required.

16.  The replication of DNA occurs in 5' to 3'
direction, simultaneously, on both strands of
DNA.
 Leading strand (continuous or forward):
 The DNA synthesis is continuous.

17.  Lagging strand (discontinuous or retrograde):
 The DNA synthesis is discontinuous, short
pieces of DNA (15-250 nucleotides) are
produced on lagging strand.
 Replication occurs in both direction from
replication bubble.

18.  The separation of two strands of parent DNA
results in the formation of replication fork.
 The active synthesis of DNA occurs in this
region.
 The replication fork moves along the parent
DNA as the daughter DNA molecules are
synthesized.

19.  DNA helicases bind to both the DNA strands
at the replication fork.
 Helicases move along the DNA helix &
separate the strands.
 Their function is comparable with a zip
opener.
 Helicases are dependent on ATP for energy
supply.

20.  Also called helix-destabilizing proteins.
 SSB proteins bind only to single-stranded DNA.
 They bind cooperatively the binding of one
molecule of SSB protein makes it easier for
additional molecules of SSB protein to bind
tightly to the DNA strand.

21.  These are not enzymes.
 These will provide single-stranded template
required by polymerases & also protects the
DNA from nucleases that degrades single-
stranded DNA.

22.  The DNA polymerases responsible for copying the
DNA templates are only able to "read" the parental
nucleotide sequences in the 3' to 5' direction & they
synthesize the new DNA strands in the 5' to 3' (anti
parallel) direction.
 The two newly synthesized nucleotide chains must
grow in opposite in the directions one in the 5' to 3'
direction toward the replication fork & one in the 5' to
3' direction away from the replication fork.

24.  Leading strand:
 The strand that is being copied in the direction
of the advancing replication fork is called the
leading strand & is synthesized continuously.
 Lagging strand:
 The strand that is being copied in the direction
away from the replication fork is synthesized
discontinuously, with small fragments of DNA
being copied near the replication fork.

25.  These short stretches of discontinuous DNA,
termed Okazaki fragments & are joined to
become a single, continuous strand.
 This is called as lagging strand.

28.  Synthesis of a new DNA strand, catalysed by
DNA polymerase lll, occurs in 5'-3' direction.
 This is antiparallel to the parent template DNA
strand.
 The presence of all the four
deoxyribonucleoside triphosphates (dATP,
dGTP, dCTP & dTTP) is an essential prerequisite
for replication to take place.

29.  The synthesis of two new DNA strands,
simultaneously, takes place in the opposite
direction - one is in a direction (5'-3') towards
the replication fork which is continuous
(Leading strand)
 The other in a direction (5'- 3') away from the
replication fork which is discontinuous
(Lagging strand).

30.  The incoming deoxyribonucleotides are
added one after another, to 3' end of the
growing DNA chain.
 A molecule of pyrophosphate (PPi) is
removed with the addition of each nucleotide.
 The template DNA strand (the parent)
determines the base sequence of the newly
synthesized complementary DNA.

31.  Prokaryotic & eukaryotic DNA polymerases
elongate a new DNA strand by adding deoxy
ribonucleotides, one at a time, to the 3'-end of
the growing chain.
 The sequence of nucleotides that are added is
dictated by the base sequence of the template
strand, with which the incoming nucleotides
are paired.

33.  The DNA strand (leading strand) with its 3'-
end (3'-OH) oriented towards the fork can be
elongated by sequential addition of new
nucleotides.
 The other DNA strand (lagging strand) with 5'-
end presents some problem,

34.  There is no DNA polymerase enzyme (in any
organism) that can catalyse the addition of
nucleotides to the 5‘ end (3'- 5' direction) of the
growing chain.
 This problem is solved by synthesizing this
strand as a series of small fragments.
 These pieces are made in the normal 5'-3'
direction & later joined together.

35.  The small fragments of the discontinuously
synthesized DNA are called Okazaki pieces.
 These are produced on the lagging strand of
the parent DNA.
 Okazaki pieces are later joined to form a
continuous strand of DNA.
 DNA polymerase I & DNA ligase are
responsible for this process.

36.  Fidelity of replication is the most important
for the very existence of an organism.
 Besides its 5'-3' directed catalytic function,
DNA polymerase III also has a proof-reading
activity.

37.  It checks the incoming nucleotides & allows
only the correctly matched bases (i.e.
complementary bases) to be added to the
growing DNA strand.
 DNA polymerase edits its mistakes (if any) &
removes the wrongly placed nucleotide
bases.

38.  For example, if the template base is cytosine
& the enzyme mistakenly inserts an adenine
instead a guanine into the new chain, the 3' to
5' exonuclease removes the misplaced
nucleotide.
 The 5' to 3' polymerase replaces it with the
correct nucleotide containing guanine.

40.  The synthesis of new DNA strand continues
till it is in close proximity to RNA primer.
 DNA polymerase I removes the RNA primer
& takes its position.
 DNA polymerase I catalyses the synthesis (5'-
3' direction) of a fragment of DNA that
replaces RNA primer.

42.  The enzyme DNA ligase catalyses the
formation of a phosphodiester linkage
between the DNA synthesized by DNA
polymerase III & the small fragments of DNA
produced by DNA polymerase l.
 This process-nick sealing-requires energy,
provided by the breakdown of ATP.
 DNA polymerase II participates in the DNA
repair process.

44.  The double helix of DNA separates from one
side & replication proceeds, supercoils are
formed at the other side.
 The problem of supercoils in DNA replication
is solved by a group of enzymes called DNA
topoisomerases.

47.  Reversibly cut a single strand of the double
helix.
 They have both nuclease (strand-cutting) &
ligase (strand-resealing) activities.
 They do not require ATP, but rather appear to
store the energy from the phosphodiester
bond they cleave, reusing the energy to reseal
the strand.

49.  Bind tightly to the DNA double helix & make
transient breaks in both strands.
 The enzyme then causes a second stretch of
the DNA double helix to pass through the
break & finally reseals the break.
 Supercoils can be relieved.

51.  Replication of DNA in eukaryotes closely
resembles that of prokaryotes.
 Certain differences exist.
 Multiple origins of replication is a
characteristic feature of eukaryotic cell.
 Five distinct DNA polymerases are known in
eukaryotes.

52.  DNA polymerase α is responsible for the
synthesis of RNA primer for both the leading
& lagging strands of DNA.
 DNA polymerase β is involved in the repair
of DNA.
 Its function is comparable with DNA
polymerasIe found in prokaryotes.

53.  DNA polymerase γ participates in the
replication of mitochondrial DNA.
 DNA polymerase δ is responsible for the
replication on the leading strand of DNA.
 It also possesses proof-reading activity.
 DNA polymerase ε is involved in DNA
synthesis on the lagging strand & proof-
reading function.

54.  The events surrounding eukaryotic DNA
replication & cell division (mitosis) are
coordinated to produce the cell cycle.
 The period preceding replication is called the
G1 phase (Gap1).
 DNA replication occurs during the S
(synthesis) phase.

55.  Following DNA synthesis, there is another
period (G2 phase, Gap2) before mitosis (M).
 Cells that have stopped dividing, such as
mature neurons, are said to have gone out of
the cell cycle into the GO phase.

57.  Bacteria contain a specific type II
topoisomerase namely gyrase.
 This enzyme cuts & reseals the circular DNA
(of bacteria) & thus overcomes the problem of
supercoils.
 Bacterial gyrase is inhibited by the antibiotics
ciprofloxacin, novobiocin & nalidixic acid.

58.  Certain compounds that inhibit human
topoisomerases are used as anticancer
agents e.g. adriamycin, etoposide,
doxorubicin.
 The nucleotide analogs that inhibit DNA
replication are also used as anticancer drugs
e.g. 6-mercaptopurnie , 5-fluorouracil.

59.  The leading strand is completely synthesized
 On lagging strand, removal of the RNA
primer leaves a small gap which cannot be
filled.
 The daughter chromosomes will have
shortened DNA molecule.
 Over a period of time, chromosomes may
lose certain essential genes & cell dies.

60.  Telomeres are the special structures that
prevent the continuous loss of DNA at the
end of the chromosomes during replication.
 Protect the ends of the chromosomes &
prevent the chromosomes from fusing with
each other.
 Human telomeres contain thousands of
repeat TTAGGG sequences, which can be up
to a length of 1500 bp.

61.  Telomerase is an unusual enzyme, it is composed of
both protein & RNA.
 In humans, RNA component is 450 nucleotides in
length, & at 5'-terminal & it contains the sequence 5‘-
CUAACCCUAAC-3'.
 Central region of this sequence is complementary to
the telomere repeat sequence 5'-TTAGGG-3'.
 Telomerase RNA sequence can be used as a
template for extension of telomere.

63.  Eukaryotic DNA is associated with tightly bound
basic proteins, called histones.
 These serve to order the DNA into basic structural
units, called nucleosomes.
 Nucleosomes are further arranged into increasingly
more complex structures that organize & condense
the long DNA molecules into chromosomes that can
be segregated during cell division.

64.  Five classes of histones -H1, H2A, H2B, H3 & H4.
 These small proteins are positively charged at
physiologic pH & contain high content of lysine
& arginine.
 They form ionic bonds with negatively
charged DNA.

66.  Two molecules each of H2A, H2B, H3 & H4 form
the structural core of the individual
nucleosome "beads.“
 Around this core, a segment of the DNA
double helix is wound nearly twice, forming
a negatively super twisted helix.
 Neighboring nucleosomes are joined by
"linker" DNA approximately fifty base pairs
long.

67.  Histone H1 of which there are several related
species, is not found in the nucleosome core,
but instead binds to the linker DNA chain
between the nucleosome beads.
 H1 is the most tissue-specific & species-specific
of the histones.
 It facilitates the packing of nucleosomes into
the more compact structures.

68.  Nucleosomes can be packed more tightly to form a
polynucleosome (also called a nucleofilament),
 This structure assumes the shape of a coil, often
referred to as a 30-nm fiber.
 The fiber is organized into loops that are anchored
by a nuclear scaffold containing several proteins.
 Additional levels of organization lead to the final
chromosomal structure.

70.  Textbook of Biochemistry - U Satyanarayana
 Textbook of Biochemistry - Lippincott’s