REPLICATION

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